Lipids and lipid nanoparticle formulations for delivery of nucleic acids

ABSTRACT

Compounds are provided having the following structure: 
                         
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein R 1 , R 2 , R 3 , L 1 , L 2 , G 1 , G 2  and G 3  are as defined herein. Use of the compounds as a component of lipid nanoparticle formulations for delivery of a therapeutic agent, compositions comprising the compounds and methods for their use and preparation are also provided.

BACKGROUND Technical Field

The present invention generally relates to novel cationic lipids thatcan be used in combination with other lipid components, such as neutrallipids, cholesterol and polymer conjugated lipids, to form lipidnanoparticles with oligonucleotides, to facilitate the intracellulardelivery of therapeutic nucleic acids (e.g. oligonucleotides, messengerRNA) both in vitro and in vivo.

Description of the Related Art

There are many challenges associated with the delivery of nucleic acidsto affect a desired response in a biological system. Nucleic acid basedtherapeutics have enormous potential but there remains a need for moreeffective delivery of nucleic acids to appropriate sites within a cellor organism in order to realize this potential. Therapeutic nucleicacids include, e.g., messenger RNA (mRNA), antisense oligonucleotides,ribozymes, DNAzymes, plasmids, immune stimulating nucleic acids,antagomir, antimir, mimic, supermir, and aptamers. Some nucleic acids,such as mRNA or plasmids, can be used to effect expression of specificcellular products as would be useful in the treatment of, for example,diseases related to a deficiency of a protein or enzyme. The therapeuticapplications of translatable nucleotide delivery are extremely broad asconstructs can be synthesized to produce any chosen protein sequence,whether or not indigenous to the system. The expression products of thenucleic acid can augment existing levels of protein, replace missing ornon-functional versions of a protein, or introduce new protein andassociated functionality in a cell or organism.

Some nucleic acids, such as miRNA inhibitors, can be used to effectexpression of specific cellular products that are regulated by miRNA aswould be useful in the treatment of, for example, diseases related todeficiency of protein or enzyme. The therapeutic applications of miRNAinhibition are extremely broad as constructs can be synthesized toinhibit one or more miRNA that would in turn regulate the expression ofmRNA products. The inhibition of endogenous miRNA can augment itsdownstream target endogenous protein expression and restore properfunction in a cell or organism as a means to treat disease associated toa specific miRNA or a group of miRNA.

Other nucleic acids can down-regulate intracellular levels of specificmRNA and, as a result, down-regulate the synthesis of the correspondingproteins through processes such as RNA interference (RNAi) orcomplementary binding of antisense RNA. The therapeutic applications ofantisense oligonucleotide and RNAi are also extremely broad, sinceoligonucleotide constructs can be synthesized with any nucleotidesequence directed against a target mRNA. Targets may include mRNAs fromnormal cells, mRNAs associated with disease-states, such as cancer, andmRNAs of infectious agents, such as viruses. To date, antisenseoligonucleotide constructs have shown the ability to specificallydown-regulate target proteins through degradation of the cognate mRNA inboth in vitro and in vivo models. In addition, antisense oligonucleotideconstructs are currently being evaluated in clinical studies.

However, two problems currently face the use of oligonucleotides intherapeutic contexts. First, free RNAs are susceptible to nucleasedigestion in plasma. Second, free RNAs have limited ability to gainaccess to the intracellular compartment where the relevant translationmachinery resides. Lipid nanoparticles formed from cationic lipids withother lipid components, such as neutral lipids, cholesterol, PEG,PEGylated lipids, and oligonucleotides have been used to blockdegradation of the RNAs in plasma and facilitate the cellular uptake ofthe oligonucleotides.

There remains a need for improved cationic lipids and lipidnanoparticles for the delivery of oligonucleotides. Preferably, theselipid nanoparticles would provide optimal drug:lipid ratios, protect thenucleic acid from degradation and clearance in serum, be suitable forsystemic or local delivery, and provide intracellular delivery of thenucleic acid. In addition, these lipid-nucleic acid particles should bewell-tolerated and provide an adequate therapeutic index, such thatpatient treatment at an effective dose of the nucleic acid is notassociated with unacceptable toxicity and/or risk to the patient. Thepresent invention provides these and related advantages.

BRIEF SUMMARY

In brief, the present invention provides lipid compounds, includingstereoisomers, pharmaceutically acceptable salts or tautomers thereof,which can be used alone or in combination with other lipid componentssuch as neutral lipids, charged lipids, steroids (including for example,all sterols) and/or their analogs, and/or polymer conjugated lipids toform lipid nanoparticles for the delivery of therapeutic agents. In someinstances, the lipid nanoparticles are used to deliver nucleic acidssuch as antisense and/or messenger RNA. Methods for use of such lipidnanoparticles for treatment of various diseases or conditions, such asthose caused by infectious entities and/or insufficiency of a protein,are also provided.

In one embodiment, compounds having the following structure (I) areprovided:

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R¹, R², R³, L¹, L², G¹, G², and G³ are as defined herein.

Pharmaceutical compositions comprising one or more of the foregoingcompounds of structure (I) and a therapeutic agent are also provided. Insome embodiments, the pharmaceutical compositions further comprise oneor more components selected from neutral lipids, charged lipids,steroids and polymer conjugated lipids. Such compositions are useful forformation of lipid nanoparticles for the delivery of the therapeuticagent.

In other embodiments, the present invention provides a method foradministering a therapeutic agent to a patient in need thereof, themethod comprising preparing a composition of lipid nanoparticlescomprising the compound of structure (I) and a therapeutic agent anddelivering the composition to the patient.

These and other aspects of the invention will be apparent upon referenceto the following detailed description.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

In the figures, identical reference numbers identify similar elements.The sizes and relative positions of elements in the figures are notnecessarily drawn to scale and some of these elements are arbitrarilyenlarged and positioned to improve figure legibility. Further, theparticular shapes of the elements as drawn are not intended to conveyany information regarding the actual shape of the particular elements,and have been solely selected for ease of recognition in the figures.

FIG. 1 shows time course of luciferase expression in mouse liver.

FIG. 2 illustrates the calculation of pKa for MC3 as a representativeexample relevant to the disclosed lipids.

FIG. 3 provides comparative luciferase activity data for selectedlipids.

DETAILED DESCRIPTION

In the following description, certain specific details are set forth inorder to provide a thorough understanding of various embodiments of theinvention. However, one skilled in the art will understand that theinvention may be practiced without these details.

The present invention is based, in part, upon the discovery of novelcationic (amino) lipids that provide advantages when used in lipidnanoparticles for the in vivo delivery of an active or therapeutic agentsuch as a nucleic acid into a cell of a mammal. In particular,embodiments of the present invention provide nucleic acid-lipidnanoparticle compositions comprising one or more of the novel cationiclipids described herein that provide increased activity of the nucleicacid and improved tolerability of the compositions in vivo, resulting ina significant increase in the therapeutic index as compared to nucleicacid-lipid nanoparticle compositions previously described.

In particular embodiments, the present invention provides novel cationiclipids that enable the formulation of improved compositions for the invitro and in vivo delivery of mRNA and/or other oligonucleotides. Insome embodiments, these improved lipid nanoparticle compositions areuseful for expression of protein encoded by mRNA. In other embodiments,these improved lipid nanoparticles compositions are useful forupregulation of endogenous protein expression by delivering miRNAinhibitors targeting one specific miRNA or a group of miRNA regulatingone target mRNA or several mRNA. In other embodiments, these improvedlipid nanoparticle compositions are useful for down-regulating (e.g.,silencing) the protein levels and/or mRNA levels of target genes. Insome other embodiments, the lipid nanoparticles are also useful fordelivery of mRNA and plasmids for expression of transgenes. In yet otherembodiments, the lipid nanoparticle compositions are useful for inducinga pharmacological effect resulting from expression of a protein, e.g.,increased production of red blood cells through the delivery of asuitable erythropoietin mRNA, or protection against infection throughdelivery of mRNA encoding for a suitable antigen or antibody.

The lipid nanoparticles and compositions of the present invention may beused for a variety of purposes, including the delivery of encapsulatedor associated (e.g., complexed) therapeutic agents such as nucleic acidsto cells, both in vitro and in vivo. Accordingly, embodiments of thepresent invention provide methods of treating or preventing diseases ordisorders in a subject in need thereof by contacting the subject with alipid nanoparticle that encapsulates or is associated with a suitabletherapeutic agent, wherein the lipid nanoparticle comprises one or moreof the novel cationic lipids described herein.

As described herein, embodiments of the lipid nanoparticles of thepresent invention are particularly useful for the delivery of nucleicacids, including, e.g., mRNA, antisense oligonucleotide, plasmid DNA,microRNA (miRNA), miRNA inhibitors (antagomirs/antimirs),messenger-RNA-interfering complementary RNA (micRNA), DNA, multivalentRNA, dicer substrate RNA, complementary DNA (cDNA), etc. Therefore, thelipid nanoparticles and compositions of the present invention may beused to induce expression of a desired protein both in vitro and in vivoby contacting cells with a lipid nanoparticle comprising one or morenovel cationic lipids described herein, wherein the lipid nanoparticleencapsulates or is associated with a nucleic acid that is expressed toproduce the desired protein (e.g., a messenger RNA or plasmid encodingthe desired protein) or inhibit processes that terminate expression ofmRNA (e.g., miRNA inhibitors). Alternatively, the lipid nanoparticlesand compositions of the present invention may be used to decrease theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid nanoparticle comprising one or more novelcationic lipids described herein, wherein the lipid nanoparticleencapsulates or is associated with a nucleic acid that reduces targetgene expression (e.g., an antisense oligonucleotide or small interferingRNA (siRNA)). The lipid nanoparticles and compositions of the presentinvention may also be used for co-delivery of different nucleic acids(e.g. mRNA and plasmid DNA) separately or in combination, such as may beuseful to provide an effect requiring colocalization of differentnucleic acids (e.g. mRNA encoding for a suitable gene modifying enzymeand DNA segment(s) for incorporation into the host genome).

Nucleic acids for use with this invention may be prepared according toany available technique. For mRNA, the primary methodology ofpreparation is, but not limited to, enzymatic synthesis (also termed invitro transcription) which currently represents the most efficientmethod to produce long sequence-specific mRNA. In vitro transcriptiondescribes a process of template-directed synthesis of RNA molecules froman engineered DNA template comprised of an upstream bacteriophagepromoter sequence (e.g. including but not limited to that from the T7,T3 and SP6 coliphage) linked to a downstream sequence encoding the geneof interest. Template DNA can be prepared for in vitro transcriptionfrom a number of sources with appropriate techniques which are wellknown in the art including, but not limited to, plasmid DNA andpolymerase chain reaction amplification (see Linpinsel, J. L and Conn,G. L., General protocols for preparation of plasmid DNA template andBowman, J. C., Azizi, B., Lenz, T. K., Ray, P., and Williams, L. D. inRNA in vitro transcription and RNA purification by denaturing PAGE inRecombinant and in vitro RNA syntheses Methods v. 941 Conn G. L. (ed),New York, N.Y.

Humana Press, 2012) Transcription of the RNA occurs in vitro using thelinearized DNA template in the presence of the corresponding RNApolymerase and adenosine, guanosine, uridine and cytidine ribonucleosidetriphosphates (rNTPs) under conditions that support polymerase activitywhile minimizing potential degradation of the resultant mRNAtranscripts. In vitro transcription can be performed using a variety ofcommercially available kits including, but not limited to RiboMax LargeScale RNA Production System (Promega), MegaScript Transcription kits(Life Technologies) as well as with commercially available reagentsincluding RNA polymerases and rNTPs. The methodology for in vitrotranscription of mRNA is well known in the art. (see, e.g. Losick, R.,1972, In vitro transcription, Ann Rev Biochem v. 41 409-46; Kamakaka, R.T. and Kraus, W. L. 2001. In Vitro Transcription. Current Protocols inCell Biology. 2:11.6:11.6.1-11.6.17; Beckert, B. And Masquida, B.,(2010) Synthesis of RNA by In Vitro Transcription in RNA in Methods inMolecular Biology v. 703 (Neilson, H. Ed), New York, N.Y. Humana Press,2010; Brunelle, J. L. and Green, R., 2013, Chapter Five—In vitrotranscription from plasmid or PCR-amplified DNA, Methods in Enzymologyv. 530, 101-114; all of which are incorporated herein by reference).

The desired in vitro transcribed mRNA is then purified from theundesired components of the transcription or associated reactions(including unincorporated rNTPs, protein enzyme, salts, short RNAoligos, etc.). Techniques for the isolation of the mRNA transcripts arewell known in the art. Well known procedures include phenol/chloroformextraction or precipitation with either alcohol (ethanol, isopropanol)in the presence of monovalent cations or lithium chloride. Additional,non-limiting examples of purification procedures which can be usedinclude size exclusion chromatography (Lukavsky, P. J. and Puglisi, J.D., 2004, Large-scale preparation and purification ofpolyacrylamide-free RNA oligonucleotides, RNA v. 10, 889-893),silica-based affinity chromatography and polyacrylamide gelelectrophoresis (Bowman, J. C., Azizi, B., Lenz, T. K., Ray, P., andWilliams, L. D. in RNA in vitro transcription and RNA purification bydenaturing PAGE in Recombinant and in vitro RNA syntheses Methods v. 941Conn G. L. (ed), New York, N.Y. Humana Press, 2012). Purification can beperformed using a variety of commercially available kits including, butnot limited to SV Total Isolation System (Promega) and In VitroTranscription Cleanup and Concentration Kit (Norgen Biotek).

Furthermore, while reverse transcription can yield large quantities ofmRNA, the products can contain a number of aberrant RNA impuritiesassociated with undesired polymerase activity which may need to beremoved from the full-length mRNA preparation. These include short RNAsthat result from abortive transcription initiation as well asdouble-stranded RNA (dsRNA) generated by RNA-dependent RNA polymeraseactivity, RNA-primed transcription from RNA templates andself-complementary 3′ extension. It has been demonstrated that thesecontaminants with dsRNA structures can lead to undesiredimmunostimulatory activity through interaction with various innateimmune sensors in eukaryotic cells that function to recognize specificnucleic acid structures and induce potent immune responses. This inturn, can dramatically reduce mRNA translation since protein synthesisis reduced during the innate cellular immune response. Therefore,additional techniques to remove these dsRNA contaminants have beendeveloped and are known in the art including but not limited toscaleable HPLC purification (see e.g. Kariko, K., Muramatsu, H., Ludwig,J. And Weissman, D., 2011, Generating the optimal mRNA for therapy: HPLCpurification eliminates immune activation and improves translation ofnucleoside-modified, protein-encoding mRNA, Nucl Acid Res, v. 39 e142;Weissman, D., Pardi, N., Muramatsu, H., and Kariko, K., HPLCPurification of in vitro transcribed long RNA in Synthetic Messenger RNAand Cell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013). HPLC purified mRNA has been reported tobe translated at much greater levels, particularly in primary cells andin vivo.

A significant variety of modifications have been described in the artwhich are used to alter specific properties of in vitro transcribedmRNA, and improve its utility. These include, but are not limited tomodifications to the 5′ and 3′ termini of the mRNA. Endogenouseukaryotic mRNA typically contain a cap structure on the 5′-end of amature molecule which plays an important role in mediating binding ofthe mRNA Cap Binding Protein (CBP), which is in turn responsible forenhancing mRNA stability in the cell and efficiency of mRNA translation.Therefore, highest levels of protein expression are achieved with cappedmRNA transcripts. The 5′-cap contains a 5′-5′-triphosphate linkagebetween the 5′-most nucleotide and guanine nucleotide. The conjugatedguanine nucleotide is methylated at the N7 position. Additionalmodifications include methylation of the ultimate and penultimate most5′-nucleotides on the 2′-hydroxyl group.

Multiple distinct cap structures can be used to generate the 5′-cap ofin vitro transcribed synthetic mRNA. 5′-capping of synthetic mRNA can beperformed co-transcriptionally with chemical cap analogs (i.e. cappingduring in vitro transcription). For example, the Anti-Reverse Cap Analog(ARCA) cap contains a 5′-5′-triphosphate guanine-guanine linkage whereone guanine contains an N7 methyl group as well as a 3′-O-methyl group.However, up to 20% of transcripts remain uncapped during thisco-transcriptional process and the synthetic cap analog is not identicalto the 5′-cap structure of an authentic cellular mRNA, potentiallyreducing translatability and cellular stability. Alternatively,synthetic mRNA molecules may also be enzymatically cappedpost-transcriptionally. These may generate a more authentic 5′-capstructure that more closely mimics, either structurally or functionally,the endogenous 5′-cap which have enhanced binding of cap bindingproteins, increased half-life, reduced susceptibility to 5′endonucleases and/or reduced 5′ decapping. Numerous synthetic 5′-capanalogs have been developed and are known in the art to enhance mRNAstability and translatability (see eg. Grudzien-Nogalska, E., Kowalska,J., Su, W., Kuhn, A. N., Slepenkov, S. V., Darynkiewicz, E., Sahin, U.,Jemielity, J., and Rhoads, R. E., Synthetic mRNAs with superiortranslation and stability properties in Synthetic Messenger RNA and CellMetabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

On the 3′-terminus, a long chain of adenine nucleotides (poly-A tail) isnormally added to mRNA molecules during RNA processing. Immediatelyafter transcription, the 3′ end of the transcript is cleaved to free a3′ hydroxyl to which poly-A polymerase adds a chain of adeninenucleotides to the RNA in a process called polyadenylation. The poly-Atail has been extensively shown to enhance both translational efficiencyand stability of mRNA (see Bernstein, P. and Ross, J., 1989, Poly (A),poly (A) binding protein and the regulation of mRNA stability, TrendsBio Sci v. 14 373-377; Guhaniyogi, J. And Brewer, G., 2001, Regulationof mRNA stability in mammalian cells, Gene, v. 265, 11-23; Dreyfus, M.And Regnier, P., 2002, The poly (A) tail of mRNAs: Bodyguard ineukaryotes, scavenger in bacteria, Cell, v. 111, 611-613).

Poly (A) tailing of in vitro transcribed mRNA can be achieved usingvarious approaches including, but not limited to, cloning of a poly (T)tract into the DNA template or by post-transcriptional addition usingPoly (A) polymerase. The first case allows in vitro transcription ofmRNA with poly (A) tails of defined length, depending on the size of thepoly (T) tract, but requires additional manipulation of the template.The latter case involves the enzymatic addition of a poly (A) tail to invitro transcribed mRNA using poly (A) polymerase which catalyzes theincorporation of adenine residues onto the 3′termini of RNA, requiringno additional manipulation of the DNA template, but results in mRNA withpoly(A) tails of heterogeneous length. 5′-capping and 3′-poly (A)tailing can be performed using a variety of commercially available kitsincluding, but not limited to Poly (A) Polymerase Tailing kit(EpiCenter), mMESSAGE mMACHINE T7 Ultra kit and Poly (A) Tailing kit(Life Technologies) as well as with commercially available reagents,various ARCA caps, Poly (A) polymerase, etc.

In addition to 5′ cap and 3′ poly adenylation, other modifications ofthe in vitro transcripts have been reported to provide benefits asrelated to efficiency of translation and stability. It is well known inthe art that pathogenic DNA and RNA can be recognized by a variety ofsensors within eukaryotes and trigger potent innate immune responses.The ability to discriminate between pathogenic and self DNA and RNA hasbeen shown to be based, at least in part, on structure and nucleosidemodifications since most nucleic acids from natural sources containmodified nucleosides In contrast, in vitro synthesized RNA lacks thesemodifications, thus rendering it immunostimulatory which in turn caninhibit effective mRNA translation as outlined above. The introductionof modified nucleosides into in vitro transcribed mRNA can be used toprevent recognition and activation of RNA sensors, thus mitigating thisundesired immunostimulatory activity and enhancing translation capacity(see e.g. Kariko, K. And Weissman, D. 2007, Naturally occurringnucleoside modifications suppress the immunostimulatory activity of RNA:implication for therapeutic RNA development, Curr Opin Drug DiscovDevel, v. 10 523-532; Pardi, N., Muramatsu, H., Weissman, D., Kariko,K., In vitro transcription of long RNA containing modified nucleosidesin Synthetic Messenger RNA and Cell Metabolism Modulation in Methods inMolecular Biology v. 969 (Rabinovich, P. H. Ed), 2013); Kariko, K.,Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., Weissman,D., 2008, Incorporation of Pseudouridine Into mRNA Yields SuperiorNonimmunogenic Vector With Increased Translational Capacity andBiological Stability, Mol Ther v. 16, 1833-1840. The modifiednucleosides and nucleotides used in the synthesis of modified RNAs canbe prepared monitored and utilized using general methods and proceduresknown in the art. A large variety of nucleoside modifications areavailable that may be incorporated alone or in combination with othermodified nucleosides to some extent into the in vitro transcribed mRNA(see e.g.US2012/0251618). In vitro synthesis of nucleoside-modified mRNAhave been reported to have reduced ability to activate immune sensorswith a concomitant enhanced translational capacity.

Other components of mRNA which can be modified to provide benefit interms of translatability and stability include the 5′ and 3′untranslated regions (UTR). Optimization of the UTRs (favorable 5′ and3′ UTRs can be obtained from cellular or viral RNAs), either both orindependently, have been shown to increase mRNA stability andtranslational efficiency of in vitro transcribed mRNA (see e.g. Pardi,N., Muramatsu, H., Weissman, D., Kariko, K., In vitro transcription oflong RNA containing modified nucleosides in Synthetic Messenger RNA andCell Metabolism Modulation in Methods in Molecular Biology v. 969(Rabinovich, P. H. Ed), 2013).

In addition to mRNA, other nucleic acid payloads may be used for thisinvention. For oligonucleotides, methods of preparation include but arenot limited to chemical synthesis and enzymatic, chemical cleavage of alonger precursor, in vitro transcription as described above, etc.Methods of synthesizing DNA and RNA nucleotides are widely used and wellknown in the art (see, e.g. Gait, M. J. (ed.) Oligonucleotide synthesis:a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press,1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods andapplications, Methods in Molecular Biology, v. 288 (Clifton, N.J.)Totowa, N.J.: Humana Press, 2005; both of which are incorporated hereinby reference).

For plasmid DNA, preparation for use with this invention commonlyutilizes but is not limited to expansion and isolation of the plasmidDNA in vitro in a liquid culture of bacteria containing the plasmid ofinterest. The presence of a gene in the plasmid of interest that encodesresistance to a particular antibiotic (penicillin, kanamycin, etc.)allows those bacteria containing the plasmid of interest to selectivelygrow in antibiotic-containing cultures. Methods of isolating plasmid DNAare widely used and well known in the art (see, e.g. Heilig, J., Elbing,K. L. and Brent, R (2001) Large-Scale Preparation of Plasmid DNA.Current Protocols in Molecular Biology. 41:II:1.7:1.7.1-1.7.16; Rozkov,A., Larsson, B., Gillstrom, S., Bjornestedt, R. and Schmidt, S. R.(2008), Large-scale production of endotoxin-free plasmids for transientexpression in mammalian cell culture. Biotechnol. Bioeng., 99: 557-566;and U.S. Pat. No. 6,197,553B1). Plasmid isolation can be performed usinga variety of commercially available kits including, but not limited toPlasmid Plus (Qiagen), GenJET plasmid MaxiPrep (Thermo) and PureYieldMaxiPrep (Promega) kits as well as with commercially available reagents.

Various exemplary embodiments of the cationic lipids of the presentinvention, lipid nanoparticles and compositions comprising the same, andtheir use to deliver active (e.g. therapeutic agents), such as nucleicacids, to modulate gene and protein expression, are described in furtherdetail below.

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

Unless the context requires otherwise, throughout the presentspecification and claims, the word “comprise” and variations thereof,such as, “comprises” and “comprising” are to be construed in an open andinclusive sense, that is, as “including, but not limited to”.

Reference throughout this specification to “one embodiment” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least oneembodiment of the present invention. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs. As used in the specification andclaims, the singular form “a”, “an” and “the” include plural referencesunless the context clearly dictates otherwise.

The phrase “induce expression of a desired protein” refers to theability of a nucleic acid to increase expression of the desired protein.To examine the extent of protein expression, a test sample (e.g. asample of cells in culture expressing the desired protein) or a testmammal (e.g. a mammal such as a human or an animal model such as arodent (e.g. mouse) or a non-human primate (e.g., monkey) model) iscontacted with a nucleic acid (e.g. nucleic acid in combination with alipid of the present invention). Expression of the desired protein inthe test sample or test animal is compared to expression of the desiredprotein in a control sample (e.g. a sample of cells in cultureexpressing the desired protein) or a control mammal (e.g., a mammal suchas a human or an animal model such as a rodent (e.g. mouse) or non-humanprimate (e.g. monkey) model) that is not contacted with or administeredthe nucleic acid. When the desired protein is present in a controlsample or a control mammal, the expression of a desired protein in acontrol sample or a control mammal may be assigned a value of 1.0. Inparticular embodiments, inducing expression of a desired protein isachieved when the ratio of desired protein expression in the test sampleor the test mammal to the level of desired protein expression in thecontrol sample or the control mammal is greater than 1, for example,about 1.1, 1.5, 2.0. 5.0 or 10.0. When a desired protein is not presentin a control sample or a control mammal, inducing expression of adesired protein is achieved when any measurable level of the desiredprotein in the test sample or the test mammal is detected. One ofordinary skill in the art will understand appropriate assays todetermine the level of protein expression in a sample, for example dotblots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, and phenotypic assays, or assaysbased on reporter proteins that can produce fluorescence or luminescenceunder appropriate conditions.

The phrase “inhibiting expression of a target gene” refers to theability of a nucleic acid to silence, reduce, or inhibit the expressionof a target gene. To examine the extent of gene silencing, a test sample(e.g. a sample of cells in culture expressing the target gene) or a testmammal (e.g. a mammal such as a human or an animal model such as arodent (e.g. mouse) or a non-human primate (e.g. monkey) model) iscontacted with a nucleic acid that silences, reduces, or inhibitsexpression of the target gene. Expression of the target gene in the testsample or test animal is compared to expression of the target gene in acontrol sample (e.g. a sample of cells in culture expressing the targetgene) or a control mammal (e.g. a mammal such as a human or an animalmodel such as a rodent (e.g. mouse) or non-human primate (e.g. monkey)model) that is not contacted with or administered the nucleic acid. Theexpression of the target gene in a control sample or a control mammalmay be assigned a value of 100%. In particular embodiments, silencing,inhibition, or reduction of expression of a target gene is achieved whenthe level of target gene expression in the test sample or the testmammal relative to the level of target gene expression in the controlsample or the control mammal is about 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Inother words, the nucleic acids are capable of silencing, reducing, orinhibiting the expression of a target gene by at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% in a test sample or a test mammal relative to thelevel of target gene expression in a control sample or a control mammalnot contacted with or administered the nucleic acid. Suitable assays fordetermining the level of target gene expression include, withoutlimitation, examination of protein or mRNA levels using techniques knownto those of skill in the art, such as, e.g., dot blots, northern blots,in situ hybridization, ELISA, immunoprecipitation, enzyme function, aswell as phenotypic assays known to those of skill in the art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as a therapeutic nucleic acid is anamount sufficient to produce the desired effect, e.g. an increase orinhibition of expression of a target sequence in comparison to thenormal expression level detected in the absence of the nucleic acid. Anincrease in expression of a target sequence is achieved when anymeasurable level is detected in the case of an expression product thatis not present in the absence of the nucleic acid. In the case where theexpression product is present at some level prior to contact with thenucleic acid, an in increase in expression is achieved when the foldincrease in value obtained with a nucleic acid such as mRNA relative tocontrol is about 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5,6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000,5000, 10000 or greater. Inhibition of expression of a target gene ortarget sequence is achieved when the value obtained with a nucleic acidsuch as antisense oligonucleotide relative to the control is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expression of atarget gene or target sequence include, e.g., examination of protein orRNA levels using techniques known to those of skill in the art such asdot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, fluorescence or luminescence ofsuitable reporter proteins, as well as phenotypic assays known to thoseof skill in the art.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of antisense molecules, plasmid DNA, cDNA, PCR products,or vectors. RNA may be in the form of small hairpin RNA (shRNA),messenger RNA (mRNA), antisense RNA, miRNA, micRNA, multivalent RNA,dicer substrate RNA or viral RNA (vRNA), and combinations thereof.Nucleic acids include nucleic acids containing known nucleotide analogsor modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, and which have similarbinding properties as the reference nucleic acid. Examples of suchanalogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, single nucleotide polymorphisms, andcomplementary sequences as well as the sequence explicitly indicated.Specifically, degenerate codon substitutions may be achieved bygenerating sequences in which the third position of one or more selected(or all) codons is substituted with mixed-base and/or deoxyinosineresidues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell.Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA)or ribose (RNA), a base, and a phosphate group. Nucleotides are linkedtogether through the phosphate groups. “Bases” include purines andpyrimidines, which further include natural compounds adenine, thymine,guanine, cytosine, uracil, inosine, and natural analogs, and syntheticderivatives of purines and pyrimidines, which include, but are notlimited to, modifications which place new reactive groups such as, butnot limited to, amines, alcohols, thiols, carboxylates, andalkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide.

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are generallycharacterized by being poorly soluble in water, but soluble in manyorganic solvents. They are usually divided into at least three classes:(1) “simple lipids,” which include fats and oils as well as waxes; (2)“compound lipids,” which include phospholipids and glycolipids; and (3)“derived lipids” such as steroids.

A “steroid” is a compound comprising the following carbon skeleton:

Non-limiting examples of steroids include cholesterol, and the like.

A “cationic lipid” refers to a lipid capable of being positivelycharged. Exemplary cationic lipids include one or more amine group(s)which bear the positive charge. Preferred cationic lipids are ionizablesuch that they can exist in a positively charged or neutral formdepending on pH. The ionization of the cationic lipid affects thesurface charge of the lipid nanoparticle under different pH conditions.This charge state can influence plasma protein absorption, bloodclearance and tissue distribution (Semple, S. C., et al., Adv. DrugDeliv Rev 32:3-17 (1998)) as well as the ability to form endosomolyticnon-bilayer structures (Hafez, I. M., et al., Gene Ther 8:1188-1196(2001)) critical to the intracellular delivery of nucleic acids.

The term “polymer conjugated lipid” refers to a molecule comprising botha lipid portion and a polymer portion. An example of a polymerconjugated lipid is a pegylated lipid. The term “pegylated lipid” refersto a molecule comprising both a lipid portion and a polyethylene glycolportion. Pegylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) andthe like.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, but are not limited to,phosphotidylcholines such as 1,2-Distearoyl-sn-glycero-3-phosphocholine(DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),phophatidylethanolamines such as1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins(SM), ceramides, steroids such as sterols and their derivatives. Neutrallipids may be synthetic or naturally derived.

The term “charged lipid” refers to any of a number of lipid species thatexist in either a positively charged or negatively charged formindependent of the pH within a useful physiological range e.g. pH ˜3 topH ˜9. Charged lipids may be synthetic or naturally derived. Examples ofcharged lipids include phosphatidylserines, phosphatidic acids,phosphatidylglycerols, phosphatidylinositols, sterol hemisuccinates,dialkyl trimethylammonium-propanes, (e.g. DOTAP, DOTMA), dialkyldimethylaminopropanes, ethyl phosphocholines, dimethylaminoethanecarbamoyl sterols (e.g. DC-Chol).

The term “lipid nanoparticle” refers to particles having at least onedimension on the order of nanometers (e.g., 1-1,000 nm) which includeone or more of the compounds of structure (I) or other specifiedcationic lipids. In some embodiments, lipid nanoparticles are includedin a formulation that can be used to deliver an active agent ortherapeutic agent, such as a nucleic acid (e.g., mRNA) to a target siteof interest (e.g., cell, tissue, organ, tumor, and the like). In someembodiments, the lipid nanoparticles of the invention comprise a nucleicacid. Such lipid nanoparticles typically comprise a compound ofstructure (I) and one or more excipient selected from neutral lipids,charged lipids, steroids and polymer conjugated lipids. In someembodiments, the active agent or therapeutic agent, such as a nucleicacid, may be encapsulated in the lipid portion of the lipid nanoparticleor an aqueous space enveloped by some or all of the lipid portion of thelipid nanoparticle, thereby protecting it from enzymatic degradation orother undesirable effects induced by the mechanisms of the host organismor cells e.g. an adverse immune response.

In various embodiments, the lipid nanoparticles have a mean diameter offrom about 30 nm to about 150 nm, from about 40 nm to about 150 nm, fromabout 50 nm to about 150 nm, from about 60 nm to about 130 nm, fromabout 70 nm to about 110 nm, from about 70 nm to about 100 nm, fromabout 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In certain embodiments, nucleic acids,when present in the lipid nanoparticles, are resistant in aqueoussolution to degradation with a nuclease. Lipid nanoparticles comprisingnucleic acids and their method of preparation are disclosed in, e.g.,U.S. Patent Publication Nos. 2004/0142025, 2007/0042031 and PCT Pub.Nos. WO 2013/016058 and WO 2013/086373, the full disclosures of whichare herein incorporated by reference in their entirety for all purposes.

As used herein, “lipid encapsulated” refers to a lipid nanoparticle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., mRNA), with full encapsulation, partial encapsulation, or both.In an embodiment, the nucleic acid (e.g., mRNA) is fully encapsulated inthe lipid nanoparticle.

As used herein, the term “aqueous solution” refers to a compositioncomprising water.

“Serum-stable” in relation to nucleic acid-lipid nanoparticles meansthat the nucleotide is not significantly degraded after exposure to aserum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of a therapeuticproduct that can result in a broad exposure of an active agent within anorganism. Some techniques of administration can lead to the systemicdelivery of certain agents, but not others. Systemic delivery means thata useful, preferably therapeutic, amount of an agent is exposed to mostparts of the body. Systemic delivery of lipid nanoparticles can be byany means known in the art including, for example, intravenous,intraarterial, subcutaneous, and intraperitoneal delivery. In someembodiments, systemic delivery of lipid nanoparticles is by intravenousdelivery.

“Local delivery,” as used herein, refers to delivery of an active agentdirectly to a target site within an organism. For example, an agent canbe locally delivered by direct injection into a disease site such as atumor, other target site such as a site of inflammation, or a targetorgan such as the liver, heart, pancreas, kidney, and the like. Localdelivery can also include topical applications or localized injectiontechniques such as intramuscular, subcutaneous or intradermal injection.Local delivery does not preclude a systemic pharmacological effect.

“Alkyl” refers to a straight or branched hydrocarbon chain radicalconsisting solely of carbon and hydrogen atoms, which is saturated orunsaturated (i.e., contains one or more double (alkenyl) and/or triplebonds (alkynyl)), having, for example, from one to twenty-four carbonatoms (C₁-C₂₄ alkyl), four to twenty carbon atoms (C₄-C₂₀ alkyl), six tosixteen carbon atoms (C₆-C₁₆ alkyl), six to nine carbon atoms (C₆-C₉alkyl), one to fifteen carbon atoms (C₁-C₁₅ alkyl), one to twelve carbonatoms (C₁-C₁₂ alkyl), one to eight carbon atoms (C₁-C₈ alkyl) or one tosix carbon atoms (C₁-C₆ alkyl) and which is attached to the rest of themolecule by a single bond, e.g., methyl, ethyl, n propyl, 1 methylethyl(iso propyl), n butyl, n pentyl, 1,1-dimethylethyl (t butyl),3-methylhexyl, 2-methylhexyl, ethenyl, prop-1-enyl, but-1-enyl,pent-1-enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl,hexynyl, and the like. Unless stated otherwise specifically in thespecification, an alkyl group is optionally substituted.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain linking the rest of the molecule to a radical group,consisting solely of carbon and hydrogen, which is saturated orunsaturated (i.e., contains one or more double (alkenylene) and/ortriple bonds (alkynylene)), and having, for example, from one totwenty-four carbon atoms (C₁-C₂₄ alkylene), one to fifteen carbon atoms(C₁-C₁₅ alkylene), one to twelve carbon atoms (C₁-C₁₂ alkylene), one toeight carbon atoms (C₁-C₈ alkylene), one to six carbon atoms (C₁-C₆alkylene), two to four carbon atoms (C₂-C₄ alkylene), one to two carbonatoms (C₁-C₂ alkylene), e.g., methylene, ethylene, propylene,n-butylene, ethenylene, propenylene, n-butenylene, propynylene,n-butynylene, and the like. The alkylene chain is attached to the restof the molecule through a single or double bond and to the radical groupthrough a single or double bond. The points of attachment of thealkylene chain to the rest of the molecule and to the radical group canbe through one carbon or any two carbons within the chain. Unless statedotherwise specifically in the specification, an alkylene chain may beoptionally substituted.

“Cycloalkyl” or “carbocyclic ring” refers to a stable non-aromaticmonocyclic or polycyclic hydrocarbon radical consisting solely of carbonand hydrogen atoms, which may include fused or bridged ring systems,having from three to fifteen carbon atoms, preferably having from threeto ten carbon atoms, and which is saturated or unsaturated and attachedto the rest of the molecule by a single bond. Monocyclic radicalsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example,adamantyl, norbornyl, decalinyl, 7,7-dimethyl-bicyclo[2.2.1]heptanyl,and the like. Unless otherwise stated specifically in the specification,a cycloalkyl group may be optionally substituted.

“Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise statedspecifically in the specification, a cycloalkylene group may beoptionally substituted.

The term “substituted” used herein means any of the above groups (e.g.alkyl, alkylene, cycloalkyl or cycloalkylene) wherein at least onehydrogen atom is replaced by a bond to a non-hydrogen atom such as, butnot limited to: a halogen atom such as F, Cl, Br, or I; oxo groups (═O);hydroxyl groups (—OH); C₁-C₁₂ alkyl groups; cycloalkyl groups;—(C═O)OR′; —O(C═O)R′; —C(═O)R′; —OR′; —S(O)_(x)R′; —S—SR′; —C(═O)SR′;—SC(═O)R′; —NR′R′; —NR′C(═O)R′; —C(═O)NR′R′; —NR′C(═O)NR′R′;—OC(═O)NR′R′; —NR′C(═O)OR′; —NR′S(O)_(x)NR′R′; —NR′S(O)_(x)R′; and—S(O)_(x)NR′R′, wherein: R′ is, at each occurrence, independently H,C₁-C₁₅ alkyl or cycloalkyl, and x is 0, 1 or 2. In some embodiments thesubstituent is a C₁-C₁₂ alkyl group. In other embodiments, thesubstituent is a cycloalkyl group. In other embodiments, the substituentis a halo group, such as fluoro. In other embodiments, the substituentis an oxo group. In other embodiments, the substituent is a hydroxylgroup. In other embodiments, the substituent is an alkoxy group (—OR′).In other embodiments, the substituent is a carboxyl group. In otherembodiments, the substituent is an amine group (—NR′R′).

“Optional” or “optionally” (e.g., optionally substituted) means that thesubsequently described event of circumstances may or may not occur, andthat the description includes instances where said event or circumstanceoccurs and instances in which it does not. For example, “optionallysubstituted alkyl” means that the alkyl radical may or may not besubstituted and that the description includes both substituted alkylradicals and alkyl radicals having no substitution.

“Prodrug” is meant to indicate a compound that may be converted underphysiological conditions or by solvolysis to a biologically activecompound of the invention. Thus, the term “prodrug” refers to ametabolic precursor of a compound of the invention that ispharmaceutically acceptable. A prodrug may be inactive when administeredto a subject in need thereof, but is converted in vivo to an activecompound of the invention. Prodrugs are typically rapidly transformed invivo to yield the parent compound of the invention, for example, byhydrolysis in blood. The prodrug compound often offers advantages ofsolubility, tissue compatibility or delayed release in a mammalianorganism (see, Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24(Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi,T., et al., A.C.S. Symposium Series, Vol. 14, and in BioreversibleCarriers in Drug Design, Ed. Edward B. Roche, American PharmaceuticalAssociation and Pergamon Press, 1987.

The term “prodrug” is also meant to include any covalently bondedcarriers, which release the active compound of the invention in vivowhen such prodrug is administered to a mammalian subject. Prodrugs of acompound of the invention may be prepared by modifying functional groupspresent in the compound of the invention in such a way that themodifications are cleaved, either in routine manipulation or in vivo, tothe parent compound of the invention. Prodrugs include compounds of theinvention wherein a hydroxy, amino or mercapto group is bonded to anygroup that, when the prodrug of the compound of the invention isadministered to a mammalian subject, cleaves to form a free hydroxy,free amino or free mercapto group, respectively. Examples of prodrugsinclude, but are not limited to, acetate, formate and benzoatederivatives of alcohol or amide derivatives of amine functional groupsin the compounds of the invention and the like.

The invention disclosed herein is also meant to encompass allpharmaceutically acceptable compounds of the compound of structure (I)being isotopically-labelled by having one or more atoms replaced by anatom having a different atomic mass or mass number. Examples of isotopesthat can be incorporated into the disclosed compounds include isotopesof hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine,and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ³¹P,³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. These radiolabeledcompounds could be useful to help determine or measure the effectivenessof the compounds, by characterizing, for example, the site or mode ofaction, or binding affinity to pharmacologically important site ofaction. Certain isotopically-labelled compounds of structure (I) or(II), for example, those incorporating a radioactive isotope, are usefulin drug and/or substrate tissue distribution studies. The radioactiveisotopes tritium, i.e., ³H, and carbon-14, i.e., ¹⁴C, are particularlyuseful for this purpose in view of their ease of incorporation and readymeans of detection.

Substitution with heavier isotopes such as deuterium, i.e., ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, for example, increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsof structure (I) can generally be prepared by conventional techniquesknown to those skilled in the art or by processes analogous to thosedescribed in the Preparations and Examples as set out below using anappropriate isotopically-labeled reagent in place of the non-labeledreagent previously employed.

The invention disclosed herein is also meant to encompass the in vivometabolic products of the disclosed compounds. Such products may resultfrom, for example, the oxidation, reduction, hydrolysis, amidation,esterification, and the like of the administered compound, primarily dueto enzymatic processes. Accordingly, the invention includes compoundsproduced by a process comprising administering a compound of thisinvention to a mammal for a period of time sufficient to yield ametabolic product thereof. Such products are typically identified byadministering a radiolabeled compound of the invention in a detectabledose to an animal, such as rat, mouse, guinea pig, monkey, or to human,allowing sufficient time for metabolism to occur, and isolating itsconversion products from the urine, blood or other biological samples.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

“Mammal” includes humans and both domestic animals such as laboratoryanimals and household pets (e.g., cats, dogs, swine, cattle, sheep,goats, horses, rabbits), and non-domestic animals such as wildlife andthe like.

“Pharmaceutically acceptable carrier, diluent or excipient” includeswithout limitation any adjuvant, carrier, excipient, glidant, sweeteningagent, diluent, preservative, dye/colorant, flavor enhancer, surfactant,wetting agent, dispersing agent, suspending agent, stabilizer, isotonicagent, solvent, or emulsifier which has been approved by the UnitedStates Food and Drug Administration as being acceptable for use inhumans or domestic animals.

“Pharmaceutically acceptable salt” includes both acid and base additionsalts.

“Pharmaceutically acceptable acid addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freebases, which are not biologically or otherwise undesirable, and whichare formed with inorganic acids such as, but are not limited to,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid and the like, and organic acids such as, but not limitedto, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid,ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid,4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid,capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid,citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonicacid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid,fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid,gluconic acid, glucuronic acid, glutamic acid, glutaric acid,2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuricacid, isobutyric acid, lactic acid, lactobionic acid, lauric acid,maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonicacid, mucic acid, naphthalene-1,5-disulfonic acid,naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid,oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid,propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid,4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid,tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroaceticacid, undecylenic acid, and the like.

“Pharmaceutically acceptable base addition salt” refers to those saltswhich retain the biological effectiveness and properties of the freeacids, which are not biologically or otherwise undesirable. These saltsare prepared from addition of an inorganic base or an organic base tothe free acid. Salts derived from inorganic bases include, but are notlimited to, the sodium, potassium, lithium, ammonium, calcium,magnesium, iron, zinc, copper, manganese, aluminum salts and the like.Preferred inorganic salts are the ammonium, sodium, potassium, calcium,and magnesium salts. Salts derived from organic bases include, but arenot limited to, salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines and basic ion exchange resins, such as ammonia,isopropylamine, trimethylamine, diethylamine, triethylamine,tripropylamine, diethanolamine, ethanolamine, deanol,2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine,lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline,betaine, benethamine, benzathine, ethylenediamine, glucosamine,methylglucamine, theobromine, triethanolamine, tromethamine, purines,piperazine, piperidine, N-ethylpiperidine, polyamine resins and thelike. Particularly preferred organic bases are isopropylamine,diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, cholineand caffeine.

Often crystallizations produce a solvate of the compound of theinvention. As used herein, the term “solvate” refers to an aggregatethat comprises one or more molecules of a compound of the invention withone or more molecules of solvent. The solvent may be water, in whichcase the solvate may be a hydrate. Alternatively, the solvent may be anorganic solvent. Thus, the compounds of the present invention may existas a hydrate, including a monohydrate, dihydrate, hemihydrate,sesquihydrate, trihydrate, tetrahydrate and the like, as well as thecorresponding solvated forms. The compound of the invention may be truesolvates, while in other cases, the compound of the invention may merelyretain adventitious water or be a mixture of water plus someadventitious solvent.

A “pharmaceutical composition” refers to a formulation of a compound ofthe invention and a medium generally accepted in the art for thedelivery of the biologically active compound to mammals, e.g., humans.Such a medium includes all pharmaceutically acceptable carriers,diluents or excipients therefor.

“Effective amount” or “therapeutically effective amount” refers to thatamount of a compound of the invention which, when administered to amammal, preferably a human, is sufficient to effect treatment in themammal, preferably a human. The amount of a lipid nanoparticle of theinvention which constitutes a “therapeutically effective amount” willvary depending on the compound, the condition and its severity, themanner of administration, and the age of the mammal to be treated, butcan be determined routinely by one of ordinary skill in the art havingregard to his own knowledge and to this disclosure.

“Treating” or “treatment” as used herein covers the treatment of thedisease or condition of interest in a mammal, preferably a human, havingthe disease or condition of interest, and includes:

(i) preventing the disease or condition from occurring in a mammal, inparticular, when such mammal is predisposed to the condition but has notyet been diagnosed as having it;

(ii) inhibiting the disease or condition, i.e., arresting itsdevelopment;

(iii) relieving the disease or condition, i.e., causing regression ofthe disease or condition; or

(iv) relieving the symptoms resulting from the disease or condition,i.e., relieving pain without addressing the underlying disease orcondition. As used herein, the terms “disease” and “condition” may beused interchangeably or may be different in that the particular maladyor condition may not have a known causative agent (so that etiology hasnot yet been worked out) and it is therefore not yet recognized as adisease but only as an undesirable condition or syndrome, wherein a moreor less specific set of symptoms have been identified by clinicians.

The compounds of the invention, or their pharmaceutically acceptablesalts may contain one or more asymmetric centers and may thus give riseto enantiomers, diastereomers, and other stereoisomeric forms that maybe defined, in terms of absolute stereochemistry, as (R)- or (S)- or, as(D)- or (L)- for amino acids. The present invention is meant to includeall such possible isomers, as well as their racemic and optically pureforms. Optically active (+) and (−), (R)- and (S)-, or (D)- and(L)-isomers may be prepared using chiral synthons or chiral reagents, orresolved using conventional techniques, for example, chromatography andfractional crystallization. Conventional techniques for thepreparation/isolation of individual enantiomers include chiral synthesisfrom a suitable optically pure precursor or resolution of the racemate(or the racemate of a salt or derivative) using, for example, chiralhigh pressure liquid chromatography (HPLC). When the compounds describedherein contain olefinic double bonds or other centers of geometricasymmetry, and unless specified otherwise, it is intended that thecompounds include both E and Z geometric isomers. Likewise, alltautomeric forms are also intended to be included.

A “stereoisomer” refers to a compound made up of the same atoms bondedby the same bonds but having different three-dimensional structures,which are not interchangeable. The present invention contemplatesvarious stereoisomers and mixtures thereof and includes “enantiomers”,which refers to two stereoisomers whose molecules are nonsuperimposeablemirror images of one another.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The present invention includestautomers of any said compounds.

Compounds

In an aspect, the invention provides novel lipid compounds which arecapable of combining with other lipid components such as neutral lipids,charged lipids, steroids and/or polymer conjugated-lipids to form lipidnanoparticles with oligonucleotides. Without wishing to be bound bytheory, it is thought that these lipid nanoparticles shieldoligonucleotides from degradation in the serum and provide for effectivedelivery of oligonucleotides to cells in vitro and in vivo.

In one embodiment, the compounds have the following structure (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomerthereof, wherein:

one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—,—C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═)NR^(a)—,—OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other of L¹ or L² is —O(C═O)—,—(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—,—NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or—NR^(a)C(═O)O— or a direct bond;

G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂alkenylene;

G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈cycloalkenylene;

R^(a) is H or C₁-C₁₂ alkyl;

R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl;

R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴;

R⁴ is C₁-C₁₂ alkyl;

R⁵ is H or C₁-C₆ alkyl; and

x is 0, 1 or 2.

In some of the foregoing embodiments, the compound has one of thefollowing structures (IA) or (IB):

wherein:

A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

R⁶ is, at each occurrence, independently H, OH or C₁-C₂₄ alkyl;

n is an integer ranging from 1 to 15.

In some of the foregoing embodiments, the compound has structure (IA),and in other embodiments, the compound has structure (IB).

In other embodiments of the foregoing, the compound has one of thefollowing structures (IC) or (ID):

wherein y and z are each independently integers ranging from 1 to 12.

In any of the foregoing embodiments, one of L¹ or L² is —O(C═O)—. Forexample, in some embodiments each of L¹ and L² are —O(C═O)—. In somedifferent embodiments of any of the foregoing, L¹ and L² are eachindependently —(C═O)O— or —O(C═O)—. For example, in some embodimentseach of L¹ and L² is —(C═O)O—.

In some different embodiments of the foregoing, the compound has one ofthe following structures (IE) or (IF):

In some of the foregoing embodiments, the compound has one of thefollowing structures (IG), (IH), (II), or (IJ):

In some of the foregoing embodiments, n is an integer ranging from 2 to12, for example from 2 to 8 or from 2 to 4. For example, in someembodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In someembodiments, n is 4. In some embodiments, n is 5. In some embodiments, nis 6.

In some other of the foregoing embodiments, y and z are eachindependently an integer ranging from 2 to 10. For example, in someembodiments, y and z are each independently an integer ranging from 4 to9 or from 4 to 6.

In some of the foregoing embodiments, R⁶ is H. In other of the foregoingembodiments, R⁶ is C₁-C₂₄ alkyl. In other embodiments, R⁶ is OH.

In some embodiments, G³ is unsubstituted. In other embodiments, G3 issubstituted. In various different embodiments, G³ is linear C₁-C₂₄alkylene or linear C₁-C₂₄ alkenylene.

In some other foregoing embodiments, R¹ or R², or both, is C₆-C₂₄alkenyl. For example, in some embodiments, R¹ and R² each, independentlyhave the following structure:

wherein:

R^(7a) and R^(7b) are, at each occurrence, independently H or C₁-C₁₂alkyl; and

a is an integer from 2 to 12,

wherein R^(7a), R^(7b) and a are each selected such that R¹ and R² eachindependently comprise from 6 to 20 carbon atoms. For example, in someembodiments a is an integer ranging from 5 to 9 or from 8 to 12.

In some of the foregoing embodiments, at least one occurrence of R^(7a)is H. For example, in some embodiments, R^(7a) is H at each occurrence.In other different embodiments of the foregoing, at least one occurrenceof R^(7b) is C₁-C₈ alkyl. For example, in some embodiments, C₁-C₈ alkylis methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl,n-hexyl or n-octyl.

In different embodiments, R¹ or R², or both, has one of the followingstructures:

In some of the foregoing embodiments, R³ is OH, CN, —C(═O)OR⁴, —OC(═O)R⁴or —NHC(═O)R⁴. In some embodiments, R⁴ is methyl or ethyl.

In various different embodiments, the compound has one of the structuresset forth in Table 1 below.

TABLE 1 Representative Compounds No. Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

It is understood that any embodiment of the compounds of structure (I),as set forth above, and any specific substituent and/or variable in thecompound structure (I), as set forth above, may be independentlycombined with other embodiments and/or substituents and/or variables ofcompounds of structure (I) to form embodiments of the inventions notspecifically set forth above. In addition, in the event that a list ofsubstituents and/or variables is listed for any particular R group, Lgroup, G group, A group, or variables a, n, x, y, or z in a particularembodiment and/or claim, it is understood that each individualsubstituent and/or variable may be deleted from the particularembodiment and/or claim and that the remaining list of substituentsand/or variables will be considered to be within the scope of theinvention.

It is understood that in the present description, combinations ofsubstituents and/or variables of the depicted formulae are permissibleonly if such contributions result in stable compounds.

In some embodiments, compositions comprising any one or more of thecompounds of structure (I) and a therapeutic agent are provided. Forexample, in some embodiments, the compositions comprise any of thecompounds of structure (I) and a therapeutic agent and one or moreexcipient selected from neutral lipids, steroids and polymer conjugatedlipids. Other pharmaceutically acceptable excipients and/or carriers arealso included in various embodiments of the compositions.

In some embodiments, the neutral lipid is selected from DSPC, DPPC,DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid isDSPC. In various embodiments, the molar ratio of the compound to theneutral lipid ranges from about 2:1 to about 8:1.

In various embodiments, the compositions further comprise a steroid orsteroid analogue. In certain embodiments, the steroid or steroidanalogue is cholesterol. In some of these embodiments, the molar ratioof the compound to cholesterol ranges from about 5:1 to 1:1.

In various embodiments, the polymer conjugated lipid is a pegylatedlipid. For example, some embodiments include a pegylated diacylglycerol(PEG-DAG) such as1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), apegylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asw-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(wo-methoxy(polyethoxy)ethyl)carbamate. Invarious embodiments, the molar ratio of the compound to the pegylatedlipid ranges from about 100:1 to about 20:1.

In some embodiments, the composition comprises a pegylated lipid havingthe following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein:

R⁸ and R⁹ are each independently a straight or branched, saturated orunsaturated alkyl chain containing from 10 to 30 carbon atoms, whereinthe alkyl chain is optionally interrupted by one or more ester bonds;and

w has a mean value ranging from 30 to 60.

In some embodiments, R⁸ and R⁹ are each independently straight,saturated alkyl chains containing from 12 to 16 carbon atoms. In someembodiments, w has a mean value ranging from 43 to 53. In otherembodiments, the average w is about 45. In other different embodiments,the average w is about 49.

In some embodiments of the foregoing composition, the therapeutic agentcomprises a nucleic acid. For example, in some embodiments, the nucleicacid is selected from antisense and messenger RNA.

In other different embodiments, the invention is directed to a methodfor administering a therapeutic agent to a patient in need thereof, themethod comprising preparing or providing any of the foregoingcompositions and administering the composition to the patient

For the purposes of administration, the compounds of the presentinvention (typically in the form of lipid nanoparticles in combinationwith a therapeutic agent) may be administered as a raw chemical or maybe formulated as pharmaceutical compositions. Pharmaceuticalcompositions of the present invention comprise a compound of structure(I) and one or more pharmaceutically acceptable carrier, diluent orexcipient. The compound of structure (I) is present in the compositionin an amount which is effective to form a lipid nanoparticle and deliverthe therapeutic agent, e.g., for treating a particular disease orcondition of interest. Appropriate concentrations and dosages can bereadily determined by one skilled in the art.

Administration of the compositions of the invention can be carried outvia any of the accepted modes of administration of agents for servingsimilar utilities. The pharmaceutical compositions of the invention maybe formulated into preparations in solid, semi-solid, liquid or gaseousforms, such as tablets, capsules, powders, granules, ointments,solutions, suspensions, suppositories, injections, inhalants, gels,microspheres, and aerosols. Typical routes of administering suchpharmaceutical compositions include, without limitation, oral, topical,transdermal, inhalation, parenteral, sublingual, buccal, rectal,vaginal, and intranasal. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intradermal,intrasternal injection or infusion techniques. Pharmaceuticalcompositions of the invention are formulated so as to allow the activeingredients contained therein to be bioavailable upon administration ofthe composition to a patient. Compositions that will be administered toa subject or patient take the form of one or more dosage units, wherefor example, a tablet may be a single dosage unit, and a container of acompound of the invention in aerosol form may hold a plurality of dosageunits. Actual methods of preparing such dosage forms are known, or willbe apparent, to those skilled in this art; for example, see Remington:The Science and Practice of Pharmacy, 20th Edition (Philadelphia Collegeof Pharmacy and Science, 2000). The composition to be administered will,in any event, contain a therapeutically effective amount of a compoundof the invention, or a pharmaceutically acceptable salt thereof, fortreatment of a disease or condition of interest in accordance with theteachings of this invention.

A pharmaceutical composition of the invention may be in the form of asolid or liquid. In one aspect, the carrier(s) are particulate, so thatthe compositions are, for example, in tablet or powder form. Thecarrier(s) may be liquid, with the compositions being, for example, anoral syrup, injectable liquid or an aerosol, which is useful in, forexample, inhalatory administration.

When intended for oral administration, the pharmaceutical composition ispreferably in either solid or liquid form, where semi-solid,semi-liquid, suspension and gel forms are included within the formsconsidered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceuticalcomposition may be formulated into a powder, granule, compressed tablet,pill, capsule, chewing gum, wafer or the like form. Such a solidcomposition will typically contain one or more inert diluents or ediblecarriers. In addition, one or more of the following may be present:binders such as carboxymethylcellulose, ethyl cellulose,microcrystalline cellulose, gum tragacanth or gelatin; excipients suchas starch, lactose or dextrins, disintegrating agents such as alginicacid, sodium alginate, Primogel, corn starch and the like; lubricantssuch as magnesium stearate or Sterotex; glidants such as colloidalsilicon dioxide; sweetening agents such as sucrose or saccharin; aflavoring agent such as peppermint, methyl salicylate or orangeflavoring; and a coloring agent.

When the pharmaceutical composition is in the form of a capsule, forexample, a gelatin capsule, it may contain, in addition to materials ofthe above type, a liquid carrier such as polyethylene glycol or oil.

The pharmaceutical composition may be in the form of a liquid, forexample, an elixir, syrup, solution, emulsion or suspension. The liquidmay be for oral administration or for delivery by injection, as twoexamples. When intended for oral administration, preferred compositioncontain, in addition to the present compounds, one or more of asweetening agent, preservatives, dye/colorant and flavor enhancer. In acomposition intended to be administered by injection, one or more of asurfactant, preservative, wetting agent, dispersing agent, suspendingagent, buffer, stabilizer and isotonic agent may be included.

The liquid pharmaceutical compositions of the invention, whether they besolutions, suspensions or other like form, may include one or more ofthe following adjuvants: sterile diluents such as water for injection,saline solution, preferably physiological saline, Ringer's solution,isotonic sodium chloride, fixed oils such as synthetic mono ordiglycerides which may serve as the solvent or suspending medium,polyethylene glycols, glycerin, propylene glycol or other solvents;antibacterial agents such as benzyl alcohol or methyl paraben;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose; agents to act as cryoprotectants such assucrose or trehalose. The parenteral preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic. Physiological saline is a preferred adjuvant. An injectablepharmaceutical composition is preferably sterile.

A liquid pharmaceutical composition of the invention intended for eitherparenteral or oral administration should contain an amount of a compoundof the invention such that a suitable dosage will be obtained.

The pharmaceutical composition of the invention may be intended fortopical administration, in which case the carrier may suitably comprisea solution, emulsion, ointment or gel base. The base, for example, maycomprise one or more of the following: petrolatum, lanolin, polyethyleneglycols, bee wax, mineral oil, diluents such as water and alcohol, andemulsifiers and stabilizers. Thickening agents may be present in apharmaceutical composition for topical administration. If intended fortransdermal administration, the composition may include a transdermalpatch or iontophoresis device.

The pharmaceutical composition of the invention may be intended forrectal administration, in the form, for example, of a suppository, whichwill melt in the rectum and release the drug. The composition for rectaladministration may contain an oleaginous base as a suitablenonirritating excipient. Such bases include, without limitation,lanolin, cocoa butter and polyethylene glycol.

The pharmaceutical composition of the invention may include variousmaterials, which modify the physical form of a solid or liquid dosageunit. For example, the composition may include materials that form acoating shell around the active ingredients. The materials that form thecoating shell are typically inert, and may be selected from, forexample, sugar, shellac, and other enteric coating agents.Alternatively, the active ingredients may be encased in a gelatincapsule.

The pharmaceutical composition of the invention in solid or liquid formmay include an agent that binds to the compound of the invention andthereby assists in the delivery of the compound. Suitable agents thatmay act in this capacity include a monoclonal or polyclonal antibody, ora protein.

The pharmaceutical composition of the invention may consist of dosageunits that can be administered as an aerosol. The term aerosol is usedto denote a variety of systems ranging from those of colloidal nature tosystems consisting of pressurized packages. Delivery may be by aliquefied or compressed gas or by a suitable pump system that dispensesthe active ingredients. Aerosols of compounds of the invention may bedelivered in single phase, bi-phasic, or tri-phasic systems in order todeliver the active ingredient(s). Delivery of the aerosol includes thenecessary container, activators, valves, subcontainers, and the like,which together may form a kit. One skilled in the art, without undueexperimentation may determine preferred aerosols.

The pharmaceutical compositions of the invention may be prepared bymethodology well known in the pharmaceutical art. For example, apharmaceutical composition intended to be administered by injection canbe prepared by combining the lipid nanoparticles of the invention withsterile, distilled water or other carrier so as to form a solution. Asurfactant may be added to facilitate the formation of a homogeneoussolution or suspension. Surfactants are compounds that non-covalentlyinteract with the compound of the invention so as to facilitatedissolution or homogeneous suspension of the compound in the aqueousdelivery system.

The compositions of the invention, or their pharmaceutically acceptablesalts, are administered in a therapeutically effective amount, whichwill vary depending upon a variety of factors including the activity ofthe specific therapeutic agent employed; the metabolic stability andlength of action of the therapeutic agent; the age, body weight, generalhealth, sex, and diet of the patient; the mode and time ofadministration; the rate of excretion; the drug combination; theseverity of the particular disorder or condition; and the subjectundergoing therapy.

Compositions of the invention may also be administered simultaneouslywith, prior to, or after administration of one or more other therapeuticagents. Such combination therapy includes administration of a singlepharmaceutical dosage formulation of a composition of the invention andone or more additional active agents, as well as administration of thecomposition of the invention and each active agent in its own separatepharmaceutical dosage formulation. For example, a composition of theinvention and the other active agent can be administered to the patienttogether in a single oral dosage composition such as a tablet orcapsule, or each agent administered in separate oral dosageformulations. Where separate dosage formulations are used, the compoundsof the invention and one or more additional active agents can beadministered at essentially the same time, i.e., concurrently, or atseparately staggered times, i.e., sequentially; combination therapy isunderstood to include all these regimens.

Preparation methods for the above compounds and compositions aredescribed herein below and/or known in the art.

It will be appreciated by those skilled in the art that in the processdescribed herein the functional groups of intermediate compounds mayneed to be protected by suitable protecting groups. Such functionalgroups include hydroxy, amino, mercapto and carboxylic acid. Suitableprotecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl(for example, t-butyldimethylsilyl, t-butyldiphenylsilyl ortrimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitableprotecting groups for amino, amidino and guanidino includet-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protectinggroups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl orarylalkyl), p-methoxybenzyl, trityl and the like. Suitable protectinggroups for carboxylic acid include alkyl, aryl or arylalkyl esters.Protecting groups may be added or removed in accordance with standardtechniques, which are known to one skilled in the art and as describedherein. The use of protecting groups is described in detail in Green, T.W. and P. G. M. Wutz, Protective Groups in Organic Synthesis (1999), 3rdEd., Wiley. As one of skill in the art would appreciate, the protectinggroup may also be a polymer resin such as a Wang resin, Rink resin or a2-chlorotrityl-chloride resin.

It will also be appreciated by those skilled in the art, although suchprotected derivatives of compounds of this invention may not possesspharmacological activity as such, they may be administered to a mammaland thereafter metabolized in the body to form compounds of theinvention which are pharmacologically active. Such derivatives maytherefore be described as “prodrugs”. All prodrugs of compounds of thisinvention are included within the scope of the invention.

Furthermore, all compounds of the invention which exist in free base oracid form can be converted to their pharmaceutically acceptable salts bytreatment with the appropriate inorganic or organic base or acid bymethods known to one skilled in the art. Salts of the compounds of theinvention can be converted to their free base or acid form by standardtechniques.

The following General Reaction Scheme 1 illustrates methods to makecompounds of this invention, i.e., compounds of structure (I):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein R¹, R², R³, L¹, L², G¹, G², and G³ are as defined herein. It isunderstood that one skilled in the art may be able to make thesecompounds by similar methods or by combining other methods known to oneskilled in the art. It is also understood that one skilled in the artwould be able to make, in a similar manner as described below, othercompounds of structure (I) not specifically illustrated below by usingthe appropriate starting components and modifying the parameters of thesynthesis as needed. In general, starting components may be obtainedfrom sources such as Sigma Aldrich, Lancaster Synthesis, Inc.,Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. orsynthesized according to sources known to those skilled in the art (see,for example, Advanced Organic Chemistry: Reactions, Mechanisms, andStructure, 5th edition (Wiley, December 2000)) or prepared as describedin this invention.

General Reaction Scheme I provides an exemplary method for preparationof compounds of structure (I). G¹, G³, R¹ and R³ in General reactionScheme 1 are as defined herein, and G1′ refers to a one-carbon shorterhomologue of G1. Compounds of structure A-1 are purchased or preparedaccording to methods known in the art. Reaction of A-1 with diol A-2under appropriate condensation conditions (e.g., DCC) yieldsester/alcohol A-3, which can then be oxidized (e.g., PCC) to aldehydeA-4. Reaction of A-4 with amine A-4 under reductive amination conditionsyields a compound of structure (I).

It should be noted that various alternative strategies for preparationof compounds of structure (I) are available to those of ordinary skillin the art. For example, other compounds of structure (I) wherein L¹ andL² are other than ester can be prepared according to analogous methodsusing the appropriate starting material. Further, General ReactionScheme 1 depicts preparation of a compound of structure (I), wherein G¹and G² are the same; however, this is not a required aspect of theinvention and modifications to the above reaction scheme are possible toyield compounds wherein G¹ and G² are different. The use of protectinggroups as needed and other modification to the above General ReactionScheme will be readily apparent to one of ordinary skill in the art.

The following examples are provided for purpose of illustration and notlimitation.

Example 1 Luciferase mRNA In Vivo Evaluation Using the LipidNanoparticle Compositions

Cationic lipid, DSPC, cholesterol and PEG-lipid were solubilized inethanol at a molar ratio of 50:10:38.5:1.5 or 47.5:10:40.8:1.7. Lipidnanoparticles (LNP) were prepared at a total lipid to mRNA weight ratioof approximately 10:1 to 30:1. Briefly, the mRNA was diluted to 0.2mg/mL in 10 to 50 mM citrate buffer, pH 4. Syringe pumps were used tomix the ethanolic lipid solution with the mRNA aqueous solution at aratio of about 1:5 to 1:3 (vol/vol) with total flow rates above 15ml/min. The ethanol was then removed and the external buffer replacedwith PBS by dialysis. Finally, the lipid nanoparticles were filteredthrough a 0.2 μm pore sterile filter. Lipid nanoparticle particle sizewas approximately 55-95 nm diameter, and in some instances approximately70-90 nm diameter as determined by quasi-elastic light scattering usinga Malvern Zetasizer Nano ZS (Malvern, UK).

Studies were performed in 6-8 week old female C57BL/6 mice (CharlesRiver) 8-10 week old CD-1 (Harlan) mice (Charles River) according toguidelines established by an institutional animal care committee (ACC)and the Canadian Council on Animal Care (CCAC). Varying doses ofmRNA-lipid nanoparticle were systemically administered by tail veininjection and animals euthanized at a specific time point (e.g 4 hrs)post-administration. Liver and spleen were collected in pre-weighedtubes, weights determined, immediately snap frozen in liquid nitrogenand stored at −80° C. until processing for analysis.

For liver, approximately 50 mg was dissected for analyses in a 2 mLFastPrep tubes (MP Biomedicals, Solon Ohio). ¼″ ceramic sphere (MPBiomedicals) was added to each tube and 500 μL of Glo Lysis Buffer—GLB(Promega, Madison Wis.) equilibrated to room temperature was added toliver tissue. Liver tissues were homogenized with the FastPrep24instrument (MP Biomedicals) at 2×6.0 m/s for 15 seconds. Homogenate wasincubated at room temperature for 5 minutes prior to a 1:4 dilution inGLB and assessed using SteadyGlo Luciferase assay system (Promega).Specifically, 50 μL of diluted tissue homogenate was reacted with 50 μLof SteadyGlo substrate, shaken for 10 seconds followed by 5 minuteincubation and then quantitated using a CentroXS³ LB 960 luminometer(Berthold Technologies, Germany). The amount of protein assayed wasdetermined by using the BCA protein assay kit (Pierce, Rockford Ill.).Relative luminescence units (RLU) were then normalized to total ugprotein assayed. To convert RLU to ng luciferase a standard curve wasgenerated with QuantiLum Recombinant Luciferase (Promega). Based in thedata provided in FIG. 1, the four-hour time point was chosen forefficacy evaluation of the lipid formulations.

The FLuc mRNA (L-6107) from Trilink Biotechnologies will express aluciferase protein, originally isolated from the firefly, Photinuspyralis. FLuc is commonly used in mammalian cell culture to measure bothgene expression and cell viability. It emits bioluminescence in thepresence of the substrate, luciferin. This capped and polyadenylatedmRNA is fully substituted with 5-methylcytidine and pseudouridine.

Example 2 Determination of pK_(a) of Formulated Lipids

As described elsewhere, the pKa of formulated cationic lipids iscorrelated with the effectiveness of LNPs for delivery of nucleic acids(see Jayaraman et al, Angewandte Chemie, International Edition (2012),51(34), 8529-8533; Semple et al, Nature Biotechnology 28, 172-176(2010)). The preferred range of pKa is ˜5 to ˜7. The pK_(a) of eachcationic lipid was determined in lipid nanoparticles using an assaybased on fluorescence of 2-(p-toluidino)-6-napthalene sulfonic acid(TNS). Lipid nanoparticles comprising of cationiclipid/DSPC/cholesterol/PEG-lipid (50/10/38.5/1.5 mol %) in PBS at aconcentration of 0.4 mM total lipid are prepared using the in-lineprocess as described in Example 1. TNS was prepared as a 100 μM stocksolution in distilled water. Vesicles were diluted to 24 μM lipid in 2mL of buffered solutions containing, 10 mM HEPES, 10 mM MES, 10 mMammonium acetate, 130 mM NaCl, where the pH ranged from 2.5 to 11. Analiquot of the TNS solution was added to give a final concentration of 1μM and following vortex mixing fluorescence intensity was measured atroom temperature in a SLM Aminco Series 2 Luminescence Spectrophotometerusing excitation and emission wavelengths of 321 nm and 445 nm. Asigmoidal best fit analysis was applied to the fluorescence data and thepK_(a) was measured as the pH giving rise to half-maximal fluorescenceintensity (see FIG. 2).

Example 3 Determination of Efficacy of Lipid Nanoparticle FormulationsContaining Various Cationic Lipids Using an In Vivo Luciferase mRNAExpression Rodent Model

The cationic lipids shown in Table 2 have previously been tested withnucleic acids. For comparative purposes, these lipids were also used toformulate lipid nanoparticles containing the FLuc mRNA (L-6107) using anin line mixing method, as described in Example 1 and in PCT/US10/22614,which is hereby incorporated by reference in its entirety. Lipidnanoparticles were formulated using the following molar ratio: 50%Cationic lipid/10% distearoylphosphatidylcholine (DSPC)/38.5%Cholesterol/1.5% PEG lipid (“PEG-DMG”, i.e.,(1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol, with anaverage PEG molecular weight of 2000). Relative activity was determinedby measuring luciferase expression in the liver 4 hours followingadministration via tail vein injection as described in Example 1. Theactivity was compared at a dose of 0.3 and 1.0 mg mRNA/kg and expressedas ng luciferase/g liver measured 4 hours after administration, asdescribed in Example 1.

TABLE 2 Comparator Lipids showing activity with mRNA Liver Luc Liver Luc@ 0.3 @ 1.0 Com- mg/kg mg/kg pound dose dose Structure MC2 4 ± 1 N/D

DLinDMA 13 ± 3  67 ± 20

MC4 41 ± 10 N/D

XTC2 80 ± 28 237 ± 99 

MC3 198 ± 126 757 ± 528

319 (2% PEG) 258 ± 67  681 ± 203

137 281 ± 203 588 ± 303

Representative compounds of the invention shown in Table 3 wereformulated using the following molar ratio: A) 50% cationic lipid/10%distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5% PEG lipid(“PEG-DMA”2-[2-(ω-methoxy(polyethyleneglycol₂₀₀₀)ethoxy]-N,N-ditetradecylacetamide)or B) 47.5% cationic lipid/10% DSPC/40.8% Cholesterol/1.7% PEG lipid.Relative activity was determined by measuring luciferase expression inthe liver 4 hours following administration via tail vein injection asdescribed in Example 1. The activity was compared at a dose of 0.3 and1.0 mg mRNA/kg and expressed as ng luciferase/g liver measured 4 hoursafter administration, as described in Example 1. A plot of selected datais given in FIG. 3 (from top to bottom: triangle=compound 3;circle=compound 2; cross=compound 1; square=MC3).

TABLE 3 Novel Cationic lipids and Associated Activity Liver Luc LiverLuc @ 0.3 mg/kg @ 1.0 mg/kg (ng luc/g (ng luc/g Lipid No. pK_(a) liver)liver) Structure Ratio 1 5.89 467 ± 72 3780 ± 210

A 2 6.05 1195 ± 245 10059 ± 3833

A 3 6.09 1275 ± 410 10643 ± 1858

A 4 5.60 378 ± 82 1952 ± 940

A 5 5.59 183 ± 45  713 ± 298

A 6 5.42 122 ± 49  520 ± 365

A 7 6.11 1158 ± 136  8406 ± 2335

A 8 5.84 1467 ± 943  7230 ± 2290

A 15 6.14 247 ± 25 1633 ± 449

A 16 6.31  344 ± 133  2633 ± 1140

A 17 6.28  275 ± 139 1554 ± 761

A 20 6.36  691 ± 150  4279 ± 2226

B 22 6.10  660 ± 184  7533 ± 4499

A 23 5.98 137 ± 51  487 ± 209

A 25 6.22 1648 ± 534 13880 ± 5083

A 26 5.84 1143 ± 782  1238 ± 1686

A 27 5.77 110 ± 42 1088 ± 802

A 30 6.09  49 ± 17 237 ± 92

A 37 5.89 1244 ± 907 2035 ± 498

A 38 6.10 60 ± 5  365 ± 181

A 44 5.79  23 ± 11  342 ± 229

B 45 6.25 1026 ± 199  8806 ± 2836

B 46 6.06  4 ± 2  5 ± 3

B

Example 4 Synthesis of 6-(2′-hexyldecanoyloxy)hexan-1-al

A solution of hexan-1,6-diol (27.6 g) in methylene chloride (475 mL) wastreated with 2-hexyldecanoic acid (19.8 g), DCC (18.2 g) and DMAP (11.3g). The solution was stirred for three days. The reaction mixture wasfiltered and hexane (500 mL) added to the filtrate. The mixture wasstirred and the precipitates allowed to settle out. The supernatant wasdecanted and washed with dilute hydrochloric acid. The organic phase wasdried over anhydrous magnesium sulfate, filtered and the solventremoved, yielding 30 g of crude product.

The crude product dissolved in methylene chloride (200 mL) and treatedwith pyridinium chlorochromate (15 g) for two hours. Diethyl ether (600mL) was added and the supernatant filtered through a silica gel bed. Thesolvent was removed from the filtrate and resultant oil dissolved inhexane. The suspension was filtered through a silica gel plug and thesolvent removed. The residue was passed down a silica gel column (80 g)using hexane, followed by methylene chloride, as the eluent.6-(2′-hexyldecanoyloxy)hexan-1-al (24 g) was obtained as a colorlessoil.

Example 5 Synthesis of 4-(2′-hexyldecanoyloxy)butan-1-al

A solution of butan-1,4-diol (12.5 g) in methylene chloride (200 mL) wastreated with 2-hexyldecanoic acid (9.2 g), DCC (8.8 g) and DMAP (4.9 g).The solution was stirred overnight. The reaction mixture was filteredand the solvent removed. The residue was dissolved in methylene chlorideand washed with dilute hydrochloric acid. The organic phase was driedover anhydrous magnesium sulfate, filtered through a silica gel bed, andthe solvent removed.

The crude product was dissolved in methylene chloride (150 mL) andtreated with pyridinium chlorochromate (6 g) for one hour. Diethyl ether(450 mL) was added and the supernatant filtered through a silica gelbed. The solvent was removed from the filtrate and resultant oildissolved in hexane. The suspension was filtered through a silica gelbed and the solvent removed, yielding 4-(2′-hexyldecanoyloxy)butan-1-al(1 g) was obtained as a colorless oil.

Example 6 Synthesis of Compound 1

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid(0.21 g) and ethanolamine (0.14 g) in methylene chloride (50 mL) wastreated with sodium triacetoxyborohydride (1.4 g) overnight. Thesolution was washed with dilute aqueous sodium hydroxide solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound 1 as a colorless oil (0.63 g).

Example 7 Synthesis of Compound 2

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid(0.33 g) and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for one hour. Thesolution was washed with dilute aqueous sodium hydroxide solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound 2 as a colorless oil (1.1 g).

Example 8 Synthesis of Compound 3

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.33 g) and 4-aminobutan-1-ol (0.23 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium bicarbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound 3 as a colorless oil (0.4 g).

Example 9 Synthesis of Compound 4

A solution of 4-(2′-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid(0.30 g) and 4-aminobutan-1-ol (0.22 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with dilute aqueous sodium hydroxide solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient. Partiallypurified fractions were passed down a second column using an aceticacid/methanol/methylene chloride (2-0/0-10/98-90%) gradient. Purefractions were washed with aqueous sodium bicarbonate solution, yieldingcompound 4 as a colorless oil (0.9 g)

Example 10 Synthesis of Compound 5

A solution of 4-(2′-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid(0.31 g) and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.4 g) for one hour. Thesolution was washed with aqueous sodium bicarbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient. Partiallypurified fractions were passed down a second column using an aceticacid/methanol/methylene chloride (2-0/0-8/98-92%) gradient. Purefractions were washed with aqueous sodium bicarbonate solution, yieldingcompound 5 as a colorless oil (0.57 g).

Example 11 Synthesis of Compound 6

A solution of 4-(2′-hexyldecanoyloxy)butan-1-al (2.4 g), acetic acid(0.30 g) and ethanolamine (0.14 g) in methylene chloride (20 mL) wastreated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-10/100-90%) gradient. Partiallypurified fractions were passed down a second column using an aceticacid/methanol/methylene chloride (2-0/0-9/98-92%) gradient. Purefractions were washed with aqueous sodium bicarbonate solution, yieldingcompound 6 as a colorless oil (0.2 g).

Example 12 Synthesis of Compound 7

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.14 g) and 5-aminopentan-1-ol (0.24 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound 7 as a colorless oil (0.5 g)

Example 13 Synthesis of Compound 8

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.17 g) and 6-aminohexan-1-ol (0.26 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) for two hours. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a methanol/methylene chloride (0-8/100-92%) gradient, yieldingcompound 8 as a colorless oil (0.5 g)

Example 14 Synthesis of Compound 9

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g) andtrans-2-aminocyclohexanol hydrochloride (0.35 g) in methylene chloride(10 mL)/tetrahydrofuran (10 mL) was treated with sodiumtriacetoxyborohydride (1.3 g) for 1.5 hours. The solution was washedwith aqueous sodium hydrogen carbonate solution. The organic phase wasdried over anhydrous magnesium sulfate, filtered and the solventremoved. The residue was passed down a silica gel column using amethanol/methylene chloride (0-8/100-92%) gradient, yielding compound 9as a colorless oil (0.6 g).

Example 15 Synthesis of Compound 10

To a solution of 2-aminoethanol (106 mg, 1.75 mmol) in any THF (15 mL),2-octyldodecyl 6-bromohexanoate (2 eq, 1.66 g, 3.5 mmol), potassiumcarbonate (2 eq, 3.5 mmol, 477 mg,) and cesium carbonate (0.3 eq, 0.525mmol, 171 mg,) were added and was heated at 63 C (oil bath) for 16 h.Trace of tetrabutylammonium iodide was added to the mixture and themixture was heated to reflux for another 4 days. The solvent wasevaporated under reduced pressure and the residue was taken in a mixtureof hexanes and ethyl acetate (ca 9:1) and washed with water and brine.The organic layer was separated and dried over anhydrous sodiumsulphate, filtered and evaporated under reduced to obtain an oil (1.6g). The residue (1.6 g) was purified by column chromatography on silicagel (MeOH in chloroform, 0 to 4%). This gave compound 10 as colorlessoil (700 mg, 0.82 mmol, 47%).

Example 16 Synthesis of Compound 11

To a solution of 2-aminoethanol (116 mg, 1.9 mmol, 115 uL) in 15 ml ofanhydrous THF, 2-hexyldecyl 6-bromohexanoate (1.9 eq, 1.52 g, 3.62mmol), potassium carbonate (1.9 eq, 3.62 mmol, 500 mg), cesium carbonate(0.3 eq, 0.57 mmol, 186 mg,) and sodium iodide (10 mg) were added andwas heated to reflux for 6 days under Ar. The solvent was evaporatedunder reduced pressure and the residue was taken up in hexanes andwashed with water and brine. The organic layer was separated, dried overanhydrous sodium sulphate, filtered and evaporated under reducedpressure to obtain a colorless oil. The crude product was purified byflash column chromatography on silica gel (MeOH in chloroform, 0 to 4%)to yield compound 11 as a colorless oil (936 mg, 1.27 mmol, 70%).

Example 17 Synthesis of Compound 12

Compound 12 was prepared in a manner analogous to the procedure forCompound 11 to yield 538 mg of colorless oil, 0.86 mmol, 57%.

Example 18 Synthesis of Compound 13

To a solution of 2-aminoethanol (171 mg, 2.81 mmol, 169 uL) in any THF(30 mL), 2-octyldodecyl 4-bromobutyrate (1.9 eq, 2.386 g, 5.33 mmol),potassium carbonate (1.9 eq, 5.33 mmol, 736 mg), cesium carbonate (0.3eq, 0.84 mmol, 275 mg) and sodium iodide (10 mg) were added and washeated to reflux for 16 h under Ar. TLC (Hexane/Ethyl acetate=9:1,CHCl₃/MeOH=19:1) showed that significant amount of 2-octyl-1-dodecanolwas produced. The mixture was cooled and filtered. The filtrate wasconcentrated and the residue was dissolved in 2-octyl-1-dodecanol (2.1g). A few beads of 4 A molecular sieves and N,N-diisopropylethylamine(1.9 equiv., 5.33 mmol, 683 mg, 0.92 mL) was added. The mixture wassealed and heated at 62 C for another 4 days. The reaction mixture wascooled. Hexane was added. The hexane solution was decanted andconcentrated to dryness. The residue was purified by by columnchromatography on silica gel (MeOH in chloroform, 0 to 4%) to yieldcompound 13 as a colorless oil (282 mg, 0.35 mmol, 13%).

Example 19 Synthesis of Compound 14

To a solution of heptadecan-9-yl 6-bromohexanoate (2 eq, 1.13 g, 2.61mmol) in any THF (15 mL), was added 2-aminoethanol (1 eq. 1.31 mmol,79.7 mg), potassium carbonate (2 eq, 2.61 mmol, 361 mg,), cesiumcarbonate (0.3 eq, 0.39 mmol, 128 mg) and sodium iodide (6 mg). Themixture was heated to reflux for 7 days under Ar. The solvent wasevaporated under reduced pressure and the residue was taken inhexanes/ethyl acetate (ca 10%) and washed with water and brine. Theorganic layer was separated and dried over anhydrous sodium sulphate,filtered and evaporated under reduced to obtain an oil (1 g). Theresidue (1 g) was purified by gravity column chromatography on silicagel (MeOH in DCM, 0 to 4%). This gave compound 14 as a colorless oil(757 mg 0.99 mmol, 76%).

Example 20 Synthesis of Compound 15

To a solution of 2-hexyldecyl 5-bromopentanoate (2 eq, 1.22 g, 3 mmol)in 15 ml of any THF (opened for 2 month), was added 4-amino-1-butanol (1eq. 1.5 mmol, 0.134 mg, 139 uL), potassium carbonate (2 eq, 3 mmol, 415mg), cesium carbonate (0.3 eq, 0.45 mmol, 146 mg) and sodium iodide (6mg). The mixture was heated to reflux for 6 days under Ar. The solventwas evaporated under reduced pressure and the residue was taken up in amixture of hexanes and ethyl acetate (ca 10%) and washed with water andbrine. The organic layer was separated and dried over anhydrous sodiumsulphate, filtered and evaporated under reduced to obtain an oil (1.12g). The residue was purified by column chromatography on silica gel(MeOH in chloroform, 0 to 5%). This gave compound 15 as colorless oil(487 mg, 0.66 mmol, 44%). ¹HNMR (400 MHz, CDCl3) δ: 5.99 (s, 1H), 3.98(d, 5.8 Hz, 4H), 3.56 (t-like, 4.8 Hz, 2H), 2.48-2.41 (m, 6H), 2.33 (t,7.4 Hz, 4H), 1.70-1.57 (m, 10H), 1.55-1.47 (m, 4H), 1.35-1.21 (48H),0.89 (t-like, 6.8 Hz, 12H).

Example 21 Synthesis of Compound 16

To a solution of 3-amino-1-propanol (0.37 mmol, 28 mg) in anhydrousacetonitrile (15 mL), 2-hexyldecyl 6-bromohexanoate (1.9 eq, 294 mg, 0.7mmol), N,N-diisopropylethylamine (2 equiv., 0.74 mmol, 96 m) and sodiumiodide (5 mg) were added and the mixture (two layers) was heated to for3 days in a pressure flask at 59° C. (oil bath). The mixture wasconcentrated and the residue was taken up in a mixture of hexane andethyl acetate (ca 5:1, 100 mL), washed with water, brine, dried oversodium sulfate, filtered and concentrated. A slightly yellow oil wasobtained (ca 300 mg). The crude product (300 mg) was purified by flashcolumn chromatography on silica gel (MeOH in chloroform, 0 to 4.4%).This gave compound 16 as colorless oil (95 mg, 0.13 mmol, 36%). ¹HNMR(400 MHz, CDCl3) δ: 5.61-5.44 (br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.80(t-like, 5.1 Hz, 2H), 2.63 (t-like, 5.6 Hz, 2H), 2.43-2.39 (m, 4H), 2.32(t, 7.5 Hz, 4H), 1.70-1.59 (m, 8H), 1.55-1.45 (m, 4H), 1.36-1.21 (52H),0.89 (t-like, 6.8 Hz, 12H).

Example 22 Synthesis of Compound 17

To a solution of 2-hexyldecyl 6-bromohexanoate (2 eq, 1.32 g, 3.14 mmol)in 15 ml of anhydrous THF, were added 4-amino-1-butanol (1 eq. 1.57mmol, 140 mg, 145 uL), potassium carbonate (2 eq, 3.14 mmol, 434 mg),cesium carbonate (0.3 eq, 0.47 mmol, 153 mg) and sodium iodide (6 mg).The mixture was heated in a pressure round-bottom flask under Ar at 75°C. (oil bath) for 6 days. The reaction mixture was cooled andconcentrated. The residue was taken up in a mixture of hexane and ethylacetate (ca 9:1), washed with water, brine, dried over sodium sulfate,filtered and concentrated to dryness (1.28 g colorless oil). The crudeproduct was purified by flash column chromatography on silica gel (MeOHin chloroform, 0 to 5%). This gave compound 17 as colorless oil (581 mg,0.76 mmol, 48%). ¹HNMR (400 MHz, CDCl3) δ: 6.43-6.17 (br. s, 1H), 3.97(d, 5.8 Hz, 4H), 3.55 (t-like, 4.7 Hz, 2H), 2.46-2.40 (m, 6H), 2.31 (t,7.5 Hz, 4H), 1.70-1.59 (m, 10H), 1.55-1.45 (m, 4H), 1.36-1.21 (52H),0.89 (t-like, 6.7 Hz, 12H).

Example 23 Synthesis of Compound 20

To a solution of 2-hexyldecyl 8-bromooctanoate (2 eq, 3.09 g, 6.9 mmol)in 30 ml of anhydrous THF, were added 4-amino-1-butanol (1 eq. 3.45mmol, 308 mg), potassium carbonate (2 eq, 6.9 mmol, 954 mg), cesiumcarbonate (0.3 eq, 1.04 mmol, 337 mg) and sodium iodide (10 mg). Themixture in a pressure round-bottom flask under Ar was heated at 64-70°C. (oil bath) for 6 days. The mixture was cooled and concentrated. Theresidue was taken up in a mixture of hexane and ethyl acetate (9:1),washed with water, brine, dried over sodium sulfate, filtered andconcentrated to dryness (colorless oil). The crude product was purifiedflash dry column chromatography on silica gel (MeOH in chloroform, 0 to4.2%). This gave compound 20 as a colorless oil (1.28 g, 1.56 mmol,45%). ¹HNMR (400 MHz, CDCl3) δ: 6.64-6.45 (br. s, 1H), 3.97 (d, 5.8 Hz,4H), 3.62-3.51 (br. 2H), 3.07-2.34 (br. 6H), 2.30 (t, 7.5 Hz, 4H),1.71-1.40 (m, 14H), 1.39-1.19 (m, 60H), 0.89 (t-like, 6.8 Hz, 12H).

Example 24 Synthesis of 9-(2′-ethylhexanoyloxy)nonan-1-al

A solution of nonan-1,9-diol (10.1 g) in methylene chloride (150 mL) wastreated with 2-ethylhexanoic acid (9.0 g), DCC (14.3 g) and DMAP (9.1g). The solution was stirred overnight. The reaction mixture wasfiltered and the solvent removed. The residue was suspended in hexaneand filtered. The filtrate was washed with dilute hydrochloric acid. Theorganic phase was dried over anhydrous magnesium sulfate, filteredthrough a silica gel bed, and the solvent removed. The crude product waspassed down a silica gel column using a methanol/methylene chloride(0-8%) gradient, to produce 9-(2′-ethylhexanoyloxy)nonan-1-ol (7.2 g) asan oil.

The 9-(2′-ethylhexanoyloxy)nonan-1-ol was dissolved in methylenechloride (100 mL) and treated with pyridinium chlorochromate (7.5 g) forone hour. Hexane (400 mL) was added and the supernatant filtered througha silica gel bed. The solvent was removed from the filtrate andresultant oil dissolved in hexane. The suspension was filtered through asilica gel bed and the solvent removed, yielding9-(2′-ethylhexanoyloxy)nonan-1-al (6 g) was obtained as a colorless oil.

Example 25 Synthesis of 9-(2′-butyloctanoyloxy)nonan-1-al

A solution of nonan-1,9-diol (12.0 g) in methylene chloride (150 mL) wastreated with 2-butyloctanoic acid (5.0 g), DCC (7.7 g) and DMAP (4.5 g).The solution was stirred overnight. The reaction mixture was filteredand the solvent removed. The residue was suspended in hexane andfiltered. The filtrate was washed with dilute hydrochloric acid. Theorganic phase was dried over anhydrous magnesium sulfate, filteredthrough a silica gel bed, and the solvent removed. The crude product waspassed down a silica gel column using a methanol/methylene chloride(0-4%) gradient, to produce 9-(2′-butyloctanoyloxy)nonan-1-ol (6 g) asan oil.

The 9-(2′-butyloctanoyloxy)nonan-1-ol was dissolved in methylenechloride (100 mL) and treated with pyridinium chlorochromate (3.8 g)overnight. Hexane (300 mL) was added and the supernatant filteredthrough a silica gel bed. The solvent was removed from the filtrate andresultant oil dissolved in hexane. The suspension was filtered through asilica gel bed and the solvent removed, yielding9-(2′-butyloctanoyloxy)nonan-1-al (3.1 g) was obtained as a colorlessoil.

Example 26 Synthesis of 6-(2′-butyloctanoyloxy)hexan-1-al

A solution of hexan-1,6-diol (9.4 g) in methylene chloride (150 mL) wastreated with 2-butyloctanoic acid (5.0 g), DCC (7.6 g) and DMAP (4.8 g).The solution was stirred overnight. The reaction mixture was filteredand the solvent removed. The residue was suspended in hexane andfiltered. The filtrate was washed with dilute hydrochloric acid. Theorganic phase was dried over anhydrous magnesium sulfate, filteredthrough a silica gel bed, and the solvent removed. The crude product waspassed down a silica gel column using a methanol/methylene chloride(0-4%) gradient, to produce 6-(2′-butyloctanoyloxy)hexan-1-ol (4.5 g) asan oil.

The 6-(2′-butyloctanoyloxy)hexan-1-ol was dissolved in methylenechloride (100 mL) and treated with pyridinium chlorochromate (4.8 g) fortwo hours. Hexane (300 mL) was added and the supernatant filteredthrough a silica gel bed. The solvent was removed from the filtrate andresultant oil dissolved in hexane. The suspension was filtered through asilica gel bed and the solvent removed, yielding6-(2′-butyloctanoyloxy)hexan-1-al (3.9 g) was obtained as a colorlessoil.

Example 27 Synthesis of 6-(2′-octyldodecanoyloxy)hexan-1-al

A solution of hexan-1,6-diol (11.5 g) in methylene chloride (150 mL)/THF(20 mL) was treated with 2-octyldodecanoic acid (9.9 g), DCC (7.5 g) andDMAP (4.7 g). The solution was stirred overnight. The reaction mixturewas filtered and the solvent removed. The residue was suspended inhexane and filtered. The filtrate was washed with dilute hydrochloricacid. The organic phase was dried over anhydrous magnesium sulfate,filtered through a silica gel bed, and the solvent removed. The crudeproduct was passed down a silica gel column using a methanol/methylenechloride (0-4%) gradient, to produce 6-(2′-octyldodecanoyloxy)hexan-1-ol(7.4 g) as an oil.

The 6-(2′-octyldodecanoyloxy)hexan-1-ol was dissolved in methylenechloride (100 mL) and treated with pyridinium chlorochromate (4.0 g) fortwo hours. Diethyl ether (300 mL) was added and the supernatant filteredthrough a silica gel bed. The solvent was removed from the filtrate andresultant oil dissolved in hexane. The suspension was filtered through asilica gel bed and the solvent removed, yielding6-(2′-octyldodecanoyloxy)hexan-1-al (5.3 g) was obtained as a colorlessoil.

Example 28 Synthesis of 6-(2′-decyltetradecanoyloxy)hexan-1-al

A solution of hexan-1,6-diol (9.6 g) in methylene chloride (150 mL) wastreated with 2-decyltetradecanoic acid (6.1 g), DCC (4.9 g) and DMAP(3.1 g). The solution was stirred overnight. The reaction mixture wasfiltered and the solvent removed. The residue was suspended in hexaneand filtered. The filtrate was washed with dilute hydrochloric acid. Theorganic phase was dried over anhydrous magnesium sulfate, filteredthrough a silica gel bed, and the solvent removed. The crude product waspassed down a silica gel column using a methanol/methylene chloride(0-4%) gradient, to produce 6-(2′-decyltetradecanoyloxy)hexan-1-ol (4.6g).

The 6-(2′-decyltetradecanoyloxy)hexan-1-ol was dissolved in methylenechloride (100 mL) and treated with pyridinium chlorochromate (3.2 g) fortwo hours. Hexane (300 mL) was added and the supernatant filteredthrough a silica gel bed. The solvent was removed from the filtrate andresultant product dissolved in hexane. The suspension was filteredthrough a silica gel bed and the solvent removed, yielding6-(2′-decyltetradecanoyloxy)hexan-1-al (4.2 g).

Example 29 Synthesis of 12-(2′-hexyldecanoyloxy)dodecan-1-al

A solution of dodecan-1,12-diol (25.0 g) in methylene chloride (300mL)/THF (100 mL) was treated with 2-hexyldecanoic acid (10.6 g), DCC(10.2 g) and DMAP (7.5 g). The solution was stirred overnight. Thereaction mixture was filtered and the solvent removed. The residue wassuspended in hexane and filtered. The filtrate was washed with water.The organic phase was dried over anhydrous magnesium sulfate, filteredthrough a silica gel bed, and the solvent removed. The crude product waspassed down a silica gel column using hexane followed by methylenechloride, to produce 12-(2′-hexyldecanoyloxy)dodecan-1-ol (7.9 g) as anoil.

The 12-(2′-hexyldecanoyloxy)dodecan-1-ol was dissolved in methylenechloride (150 mL) and treated with pyridinium chlorochromate (4.0 g) forthree hours. Hexane (300 mL) was added and the supernatant filteredthrough a silica gel bed. The solvent was removed from the filtrate andresultant oil dissolved in hexane. The suspension was filtered through asilica gel bed and the solvent removed, yielding12-(2′-hexyldecanoyloxy)dodecan-1-al (3.9 g) was obtained as a colorlessoil.

Example 30 Synthesis of 9-(2′-hexyldecanoyloxy)nonan-1-al

A solution of nonan-1,9-diol (46.8 g) in methylene chloride (600 mL) wastreated with 2-hexyldecanoic acid (25.0 g), DCC (22.0 g) and DMAP (15.0g). The solution was stirred overnight. The reaction mixture wasfiltered and the solvent removed. The residue was suspended in hexaneand filtered. The filtrate was washed with dilute hydrochloric acid. Theorganic phase was dried over anhydrous magnesium sulfate, filteredthrough a silica gel bed, and the solvent removed. The crude product waspassed down a silica gel column using hexane followed by amethanol/methylene chloride (0-8%) gradient, to produce9-(2′-hexyldecanoyloxy)nonan-1-ol (22 g) as an oil.

9-(2′-Hexyldecanoyloxy)nonan-1-ol (5.0 g) was dissolved in methylenechloride (50 mL) and treated with pyridinium chlorochromate (2.7 g) forone hour. Hexane (200 mL) was added and the supernatant filtered througha silica gel bed. The solvent was removed from the filtrate andresultant oil dissolved in hexane. The suspension was filtered through asilica gel bed and the solvent removed, yielding9-(2′-hexyldecanoyloxy)nonan-1-al (3.6 g) was obtained as a colorlessoil.

Example 31 Synthesis of Compound 22

A solution of 9-(2′-hexyldecanoyloxy)nonan-1-al (2.2 g), acetic acid(0.15 g) and 4-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.30 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-12/98-88%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 22 as a colorless oil(0.93 g).

Example 32 Synthesis of Compound 23

A solution of 12-(2′-hexyldecanoyloxy)dodecan-1-al (2.0 g), acetic acid(0.09 g) and 4-aminobutan-1-ol (0.14 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (0.71 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-12/98-88%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 23 as a colorless oil(1.0 g).

Example 33 Synthesis of Compound 24

A solution of 9-(2′-ethylhexanoyloxy)nonan-1-al (3.0 g), acetic acid(0.11 g) and 4-aminobutan-1-ol (0.17 g) in methylene chloride (50 mL)was treated with sodium triacetoxyborohydride (0.89 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-10/98-90%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 24 as a colorless oil(0.69 g).

Example 34 Synthesis of Compound 25

A solution of 9-(2′-butyloctanoyloxy)nonan-1-al (2.6 g), acetic acid(0.20 g) and 4-aminobutan-1-ol (0.26 g) in methylene chloride (50 mL)was treated with sodium triacetoxyborohydride (1.42 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-12/98-88%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 25 as a colorless oil(0.82 g).

Example 35 Synthesis of Compound 26

A solution of 6-(2′-octyldodecanoyloxy)hexan-1-al (2.7 g), acetic acid(0.20 g) and 4-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.30 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-12/98-88%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 26 as a colorless oil(0.21 g).

Example 36 Synthesis of Compound 27

A solution of 6-(2′-decyltetradecanoyloxy)hexan-1-al (2.1 g), aceticacid (0.11 g) and 4-aminobutan-1-ol (0.13 g) in methylene chloride (30mL) was treated with sodium triacetoxyborohydride (0.70 g) overnight.The solution was washed with aqueous sodium hydrogen carbonate solution.The organic phase was dried over anhydrous magnesium sulfate, filteredand the solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-12/98-88%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 27 as a colorless oil(0.90 g).

Example 37 Synthesis of Compound 28

A solution of 6-(2′-butyloctanoyloxy)hexan-1-al (2.0 g), acetic acid(0.13 g) and 3-aminopropan-1-ol (0.13 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.0 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-8/98-92%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 28 as a colorless oil(0.77 g).

Example 38 Synthesis of Compound 30

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.15 g) and 3-aminopropan-1,2-diol (0.21 g) in methylene chloride (20mL) was treated with sodium triacetoxyborohydride (1.76 g) overnight.The solution was washed with aqueous sodium hydrogen carbonate solution.The organic phase was dried over anhydrous magnesium sulfate, filteredand the solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-12/98-88%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 30 as a colorless oil(0.60 g).

Example 39 Synthesis of Compound 31

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.15 g) and 2-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.1 g) for two hours. Thesolution was washed with dilute aqueous sodium hydroxide solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-4/98-96%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 31 as a colorless oil(0.31 g).

Example 40 Synthesis of Compound 37

A solution of 6-(2′-octyldodecanoyloxy)hexan-1-al (2.7 g), acetic acid(0.20 g) and 3-aminopropan-1-ol (0.17 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.3 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-12/98-88%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 37 as a colorless oil(0.22 g).

Example 41 Synthesis of Compound 38

A solution of 12-(2′-hexyldecanoyloxy)dodecan-1-al (1.8 g), acetic acid(0.08 g) and 3-aminopropan-1-ol (0.11 g) in methylene chloride (10 mL)was treated with sodium triacetoxyborohydride (0.64 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-10/98-90%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 38 as a colorless oil(0.83 g).

Example 42 Synthesis of Compound 39

A mixture (two layers) of ethyl 4-aminobutyrate hydrochloride salt (1.28mmol, 214 mg), 2-hexyldecyl 6-bromohexanoate (1.9 eq, 2.43 mmol, 1.02g), N,N-diisopropylethylamine (3.5 equiv., 4.48 mmol, 579 mg) and sodiumiodide (5 mg) in anhydrous acetonitrile (15 mL) was heated at 60° C. for2 days in a pressure flask. The mixture was cooled and concentrated. Theresidue was taken up in a mixture of hexane and ethyl acetate (ca 5:1,100 mL), washed with water, brine, dried over sodium sulfate, filteredand concentrated. A brown oil was obtained (ca 1.04 g). The crudeproduct was purified by flash column chromatography on silica gel (MeOHin DCM, 0 to 3.5%). This gave compound 39 as a colorless oil (334 mg,0.41 mmol, 43%). ¹HNMR (400 MHz, CDCl3) δ: 4.13 (q, 7.1 Hz, 2H), 3.97(d, 5.8 Hz, 4H), 2.43-2.34 (m, 6H), 2.33-2.28 (m, 6H), 1.73 (quintet,7.3 Hz, 2H), 1.68-1.58 (m, 6H), 1.47-1.37 (m, 4H), 1.36-1.20 (54H), 0.89(t-like, 6.8 Hz, 12H).

Example 43 Synthesis of Compound 40

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.15 g) and 1-aminobutan-2-ol (0.10 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.8 g) for two hours. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-8/98-92%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 40 as a colorless oil(0.85 g).

Example 44 Synthesis of Compound 41

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (2.4 g), acetic acid(0.19 g) and 3-methoxypropylamine (0.21 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.8 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-8/98-92%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 41 as a colorless oil(0.77 g).

Example 45 Synthesis of Compound 42

A solution of 6-(2′-butyloctanoyloxy)hexan-1-al (2.0 g), acetic acid(0.13 g) and 4-aminobutan-1-ol (0.20 g) in methylene chloride (20 mL)was treated with sodium triacetoxyborohydride (1.03 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-8/98-92%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 42 as a colorless oil(0.54 g).

Example 46 Synthesis of Compound 43

A solution of 9-(2′-ethylhexanoyloxy)nonan-1-al (3.0 g), acetic acid(0.11 g) and 3-aminopropan-1-ol (0.14 g) in methylene chloride (50 mL)was treated with sodium triacetoxyborohydride (0.91 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-6/98-94%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 43 as a colorless oil(1.01 g).

Example 47 Synthesis of Compound 44

A solution of 6-(2′-decyltetradecanoyloxy)hexan-1-al (2.1 g), aceticacid (0.11 g) and 3-aminopropan-1-ol (0.11 g) in methylene chloride (30mL) was treated with sodium triacetoxyborohydride (0.71 g) overnight.The solution was washed with aqueous sodium hydrogen carbonate solution.The organic phase was dried over anhydrous magnesium sulfate, filteredand the solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-8/98-96%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 44 as a colorless oil(1.07 g).

Example 48 Synthesis of Compound 45

A solution of 9-(2′-butyloctanoyloxy)nonan-1-al (2.6 g), acetic acid(0.17 g) and 3-aminopropan-1-ol (0.21 g) in methylene chloride (50 mL)was treated with sodium triacetoxyborohydride (1.34 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-8/98-96%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 45 as a colorless oil(1.1 g).

Example 49 Synthesis of Compound 46

To a solution of 2-aminoethanol (96.5 mg, 1.58 mmol, 95.4 uL, MW 61.08,d 1.012) in 15 ml of 2-propanol, 2-hexyldecyl 8-bromooctanoate (1.8 eq,1.27 g, 2.84 mmol), potassium carbonate (1.9 eq, 3 mmol, 414 mg), cesiumcarbonate (0.3 eq, 0.47 mmol, 154 mg) and sodium iodide (10 mg) wereadded and was heated for 3 days (oil bath 60° C.). The mixture wasconcentrated and the residue was taken up in THF (10 mL). To thismixture more aminoethanol (80 mg, 1.3 mmol) was added. Heating wascontinued at 70° C. for another 3 days. After total 6 days, the reactionmixture was cooled and filtered and concentrated. The residue waspurified flash dry column chromatography on silica gel (methanol inchloroform, 1 to 4.2%). This gave compound 46 as a colorless oil (334mg, 0.42 mmol, 30%). ¹HNMR (400 MHz, CDCl3) δ: 4.09-4.06 (m, 2H), 3.97(d, 5.8 Hz, 4H), 3.39-3.36 (m, 2H), 3.31-3.23 (m, 4H), 2.31 (t, 7.5 Hz,4H), 1.88-1.56 (m, 12H), 1.43-1.19 (59H), 0.89 (t-like, 6.8 Hz, 12H).

Example 50 Synthesis of Compound 47

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (3.0 g), acetic acid(0.20 g) and 3-aminopropionitrile (0.21 g) in methylene chloride (30 mL)was treated with sodium triacetoxyborohydride (1.3 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-6/98-94%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 47 as a colorless oil(0.29 g).

Example 51 Synthesis of Compound 48

A solution of 6-(2′-hexyldecanoyloxy)hexan-1-al (3.0 g) and ethyl4-aminobutyrate hydrochloride (0.46 g) in methylene chloride (30 mL) wastreated with sodium triacetoxyborohydride (1.4 g) overnight. Thesolution was washed with aqueous sodium hydrogen carbonate solution. Theorganic phase was dried over anhydrous magnesium sulfate, filtered andthe solvent removed. The residue was passed down a silica gel columnusing a using an acetic acid/methanol/methylene chloride(2-0/0-8/98-92%) gradient. Pure fractions were washed with aqueoussodium bicarbonate solution, yielding compound 48 as a colorless oil(0.80 g).

Example 52 Synthesis of Compound 49

To a solution of 2-butyloctyl 8-bromooctanoate (2 eq, 1.877 g, 4.8 mmol)in 20 ml of anhydrous THF, were added 4-amino-1-butanol (1 eq. 2.4 mmol,214 mg, 221 ul), potassium carbonate (2 eq, 4.8 mmol, 664 mg), cesiumcarbonate (0.3 eq, 0.72 mmol, 234 mg) and sodium iodide (ca 5 mg). Themixture in a pressure round-bottom flask was heated (oil bath, 80° C.)for 6 days. The reaction mixture was cooled and concentrated. Theresidue was taken up in a mixture of hexane and ethyl acetate (ca 5:1),washed with water, brine, dried over sodium sulfate, filtered andconcentrated. The residue was purified flash column chromatography onsilica gel (methanol in chloroform, 1 to 4%). This gave compound 49 as acolorless oil (857 mg, 1.21 mmol, 50%). ¹HNMR (400 MHz, CDCl3) δ: 6.55(br. s, 1H), 3.97 (d, 5.8 Hz, 4H), 3.55 (not well resolved triplet, 2H),2.45-2.40 (m. 6H), 2.30 (t, 7.5 Hz, 4H), 1.71-1.58 (m, 10H), 1.51-1.42(m, 4H), 1.39-1.19 (m, 44H), 0.93-0.87 (m, 12H).

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, U.S. patent applications, foreign patents, foreign patentapplications and non-patent publications referred to in thisspecification and/or listed in the Application Data Sheet areincorporated herein by reference, in their entirety. Aspects of theembodiments can be modified, if necessary to employ concepts of thevarious patents, applications and publications to provide yet furtherembodiments. These and other changes can be made to the embodiments inlight of the above-detailed description. In general, in the followingclaims, the terms used should not be construed to limit the claims tothe specific embodiments disclosed in the specification and the claims,but should be construed to include all possible embodiments along withthe full scope of equivalents to which such claims are entitled.Accordingly, the claims are not limited by the disclosure.

The invention claimed is:
 1. A compound having the following structure(IF):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:G¹ and G² are each independently unsubstituted C₄-C₁₂ alkylene or C₄-C₁₂alkenylene; G³ is C₃-C₆ alkylene; R¹ and R² are each independentlybranched C₆-C₂₄ alkyl; R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or—NR⁵C(═O)R⁴; R⁴ is C₁-C₁₂ alkyl; and R⁵ is H or C₁-C₆ alkyl.
 2. Thecompound of claim 1, having the following structures (IH):

wherein: R⁶ is H, OH or C₁-C₂₄ alkyl; y and z are each independentlyintegers ranging from 4 to 12; and n is an integer ranging from 3 to 6.3. The compound of claim 2, wherein n is
 3. 4. The compound of claim 2,wherein n is.
 5. The compound of claim 2, wherein y and z are eachindependently an integer ranging from 4 to
 9. 6. The compound of claim1, wherein R¹ and R² each, independently have the following structure:

wherein: R^(7a) and R^(7b) are, at each occurrence, independently H orC₁-C₁₂ alkyl; and a is an integer from 2 to 12, wherein R^(7a), R^(7b)and a are each selected such that R¹ and R² are each independently abranched alkyl comprising from 6 to 20 carbon atoms.
 7. The compound ofclaim 6, wherein a is an integer from 8 to
 12. 8. The compound of claim6, wherein at least one occurrence of R^(7a) is H.
 9. The compound ofclaim 6, wherein R^(7a) is H at each occurrence.
 10. The compound ofclaim 6, wherein at least one occurrence of R^(7b) is C₁-C₈ alkyl. 11.The compound of claim 10, wherein C₁-C₈ alkyl is methyl, ethyl,n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl orn-octyl.
 12. The compound of claim 1, wherein R¹ or R², or both, has oneof the following structures:


13. The compound of claim 1, wherein R³ is OH.
 14. The compound of claim1, wherein R³ is CN.
 15. The compound of claim 1, wherein R³ is—C(═O)OR⁴, —OC(═O)R⁴ or —NHC(═O)R⁴.
 16. The compound of claim 15,wherein R⁴ is methyl or ethyl.
 17. The compound of claim 1, having oneof the following structures:


18. A composition comprising the compound of claim 1 and a therapeuticagent comprising a nucleic acid.
 19. The composition of claim 18,further comprising one or more excipient selected from neutral lipids,steroids and polymer conjugated lipids.
 20. The composition of claim 19,wherein the composition comprises one or more neutral lipids selectedfrom DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.
 21. The composition ofclaim 20, wherein the neutral lipid is DSPC.
 22. The composition ofclaim 18, wherein the molar ratio of the compound to the neutral lipidranges from about 2:1 to about 8:1.
 23. The composition of claim 19,wherein the steroid is cholesterol.
 24. The composition of claim 23,wherein the molar ratio of the compound to cholesterol ranges from 5:1to 1:1.
 25. The composition of claim 19, wherein the polymer conjugatedlipid is a pegylated lipid.
 26. The composition of claim 25, wherein themolar ratio of the compound to the pegylated lipid ranges from about100:1 to about 20:1.
 27. The composition of claim 25, wherein thepegylated lipid is PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer or a PEGdialkyoxypropylcarbamate.
 28. The composition of claim 25, wherein thepegylated lipid has the following structure (II):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof,wherein: R⁸ and R⁹ are each independently a straight or branched,saturated or unsaturated alkyl chain containing from 10 to 30 carbonatoms, wherein the alkyl chain is optionally interrupted by one or moreester bonds; and w has a mean value ranging from 30 to
 60. 29. Thecomposition of claim 28, wherein R⁸ and R⁹ are each independentlystraight, saturated alkyl chains containing from 12 to 16 carbon atoms.30. The composition of claim 28, wherein the average w is about
 49. 31.The composition of claim 18, wherein the nucleic acid is selected fromantisense and messenger RNA.
 32. A method for administering atherapeutic agent comprising a nucleic acid to a patient in needthereof, the method comprising administering the composition of claim 18to the patient.
 33. A lipid nanoparticle comprising the compound ofclaim 1 and a therapeutic agent comprising a nucleic acid.
 34. The lipidnanoparticle of claim 33, wherein the therapeutic agent is selected fromantisense and messenger RNA.